Cloning of gene-encoded stem bromelain on system coming from Pichia pastoris as therapeutic protein candidate

NASA Astrophysics Data System (ADS)

Yusuf, Y.; Hidayati, W.

2018-01-01

The process of identifying bacterial recombination using PCR, and restriction, and then sequencing process was done after identifying the bacteria. This research aimed to get a yeast cell of Pichia pastoris which has an encoder gene of stem bromelain enzyme. The production of recombinant stem bromelain enzymes using yeast cells of P. pastoris can produce pure bromelain rod enzymes and have the same conformation with the enzyme’s conformation in pineapple plants. This recombinant stem bromelain enzyme can be used as a therapeutic protein in inflammatory, cancer and degenerative diseases. This study was an early stage of a step series to obtain bromelain rod protein derived from pineapple made with genetic engineering techniques. This research was started by isolating the RNA of pineapple stem which was continued with constructing cDNA using reserve transcriptase-PCR technique (RT-PCR), doing the amplification of bromelain enzyme encoder gene with PCR technique using a specific premiere couple which was designed. The process was continued by cloning into bacterium cells of Escherichia coli. A vector which brought the encoder gene of stem bromelain enzyme was inserted into the yeast cell of P. pastoris and was continued by identifying the yeast cell of P. pastoris which brought the encoder gene of stem bromelain enzyme. The research has not found enzyme gene of stem bromelain in yeast cell of P. pastoris yet. The next step is repeating the process by buying new reagent; RNase inhibitor, and buying liquid nitrogen.

The MdTFL1 gene of apple (Malus x domestica Borkh.) reduces vegetative growth and generation time.

PubMed

Flachowsky, Henryk; Szankowski, Iris; Waidmann, Sascha; Peil, Andreas; Tränkner, Conny; Hanke, Magda-Viola

2012-10-01

TFL1 is known as a floral repressor in Arabidopsis thaliana (L.) Heynh. In apple there are two TFL1 homologs, MdTFL1-1 and MdTFL1-2. The MdTFL1-1 gene was silenced in transgenic clones expressing a hairpin gene construct of a 323 bp fragment of MdTFL1-1. The hairpin gene construct was transferred to three different apple genotypes. Of 22 transgenic clones, 21 showed a significant reduction in MdTFL1-1 mRNA expression. Precocious flowering was obtained for 20 clones, which flowered already during in vitro cultivation. Nineteen clones could successfully be transferred to the greenhouse where 18 of them flowered within a few weeks followed by the death or at least a strongly inhibited vegetative growth of the plant. Most of the transgenic flowers developed abnormally. Results obtained on greenhouse-grown plants of the transgenic clones and transgenic seedlings clearly demonstrated the major role of MdTFL1 genes in maintaining the vegetative growth as prerequisite for a perennial lifecycle. It was shown that MdTFL1 dsRNAi promotes a life history similar to annual plants. Preliminary results obtained from grafting experiments with non-transgenic scions grafted onto MdTFL1 dsRNAi transgenic rootstocks indicated that the flower-inducing signal obtained after silencing of MdTFL1 genes seems not to be graft-transmissible.

Post-mortem re-cloning of a transgenic red fluorescent protein dog.

PubMed

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

2011-12-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

Expression of EGFP and NPTII protein is not associated with organ abnormalities in deceased transgenic cloned cattle.

PubMed

Liu, Yan; Wu, Qian; Cui, Huiting; Li, Qinghe; Zhao, Yiqiang; Luo, Juan; Liu, Qiuyue; Sun, Xiuzhu; Tang, Bo; Zhang, Lei; Dai, Yunping; Li, Ning

2008-12-01

Both enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase type II enzyme (NPTII) are widely used in transgenic studies, but their side effects have not been extensively investigated. In this study, we evaluated the expression profiles of the two marker genes and the relationship between their expression and organ abnormalities. Eight transgenic cloned cattle were studied, four harboring both EGFP and NPTII, and four harboring only the NPTII gene. Four age-matched cloned cattle were used as controls. EGFP and NPTII expression were measured and detected by Q-PCR, Western blot, ELISA, and RIA in heart, liver, and lungs, and the values ranged from 0.3 to 5 microg/g. The expression profiles exhibited differential or mosaic pattern between the organs, the pathologic symptoms of which were identified, but were similar to those of age-matched cloned cattle. All data indicated that the expression of EGFP and NPTII is not associated with organ abnormalities in transgenic cloned cattle.

Enhanced iron and zinc accumulation in genetically engineered pineapple plants using soybean ferritin gene.

PubMed

Mhatre, Minal; Srinivas, Lingam; Ganapathi, Thumballi R

2011-12-01

Pineapple (Ananas comosus L. Merr., cv. "Queen") leaf bases were transformed with Agrobacterium tumefaciens strain EHA 105 harboring the pSF and pEFESF plasmids with soybean ferritin cDNA. Four to eight percent of the co-cultivated leaf bases produced multiple shoots 6 weeks after transfer to Murashige and Skoog's medium supplemented with α-naphthalene acetic acid 1.8 mg/l, indole-3-butyric acid 2.0 mg/l, kinetin 2.0 mg/l, cefotaxime 400 mg/l, and kanamycin 50 mg/l. Putatively transformed shoots (1-2 cm) were selected and multiplied on medium of the same composition and elongated shoots (5 cm) were rooted on liquid rooting medium supplemented with cefotaxime 400 mg/l and kanamycin 100 mg/l. The rooted plants were analyzed through PCR, genomic Southern analysis, and reverse transcription PCR. The results clearly confirmed the integration and expression of soybean ferritin gene in the transformed plants. Atomic absorption spectroscopic analysis carried out with six independently transformed lines of pSF and pEFE-SF revealed a maximum of 5.03-fold increase in iron and 2.44-fold increase in zinc accumulation in the leaves of pSF-transformed plants. In pEFE-SF-transformed plants, a 3.65-fold increase in iron and 2.05-fold increase in zinc levels was observed. Few of the transgenic plants were hardened in the greenhouse and are being grown to maturity to determine the enhanced iron and zinc accumulation in the fruits. To the best of our knowledge this is the first report on the transformation of pineapple with soybean ferritin for enhanced accumulation of iron and zinc content in the transgenic plants.

Recloned dogs derived from adipose stem cells of a transgenic cloned beagle.

PubMed

Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Hong, So Gun; Ra, Jeong Chan; Jo, Jung Youn; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2011-04-15

A number of studies have postulated that efficiency in mammalian cloning is inversely correlated with donor cell differentiation status and may be increased by using undifferentiated cells as nuclear donors. Here, we attempted the recloning of dogs by nuclear transfer of canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) from a transgenic cloned beagle to determine if cAd-MSCs can be a suitable donor cell type. In order to isolate cAd-MSCs, adipose tissues were collected from a transgenic cloned beagle produced by somatic cell nuclear transfer (SCNT) of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. The cAd-MSCs expressed the RFP gene and cell-surface marker characteristics of MSCs including CD29, CD44 and thy1.1. Furthermore, cAd-MSCs underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. In order to investigate the developmental potential of cAd-MSCs, we carried out SCNT. Fused-couplets (82/109, 75.2%) were chemically activated and transferred into the uterine tube of five naturally estrus-synchronized surrogates. One of them (20%) maintained pregnancy and subsequently gave birth to two healthy cloned pups. The present study demonstrated for the first time the successful production of cloned beagles by nuclear transfer of cAd-MSCs. Another important outcome of the present study is the successful recloning of RFP-expressing transgenic cloned beagle pups by nuclear transfer of cells derived from a transgenic cloned beagle. In conclusion, the present study demonstrates that adipose stem cells can be a good nuclear donor source for dog cloning. Copyright © 2011 Elsevier Inc. All rights reserved.

Post-mortem re-cloning of a transgenic red fluorescent protein dog

PubMed Central

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo

2011-01-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification. PMID:22122908

High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow.

PubMed

Salamone, Daniel; Barañao, Lino; Santos, Claudio; Bussmann, Leonardo; Artuso, Jorge; Werning, Carlos; Prync, Aida; Carbonetto, Cesar; Dabsys, Susana; Munar, Carlos; Salaberry, Roberto; Berra, Guillermo; Berra, Ignacio; Fernández, Nahuel; Papouchado, Mariana; Foti, Marcelo; Judewicz, Norberto; Mujica, Ignacio; Muñoz, Luciana; Alvarez, Silvina Fenández; González, Eliseo; Zimmermann, Juan; Criscuolo, Marcelo; Melo, Carlos

2006-07-13

Transgenic farm animals have been proposed as an alternative to current bioreactors for large scale production of biopharmaceuticals. However, the efficiency of both methods in the production of the same protein has not yet been established. Here we report the production of recombinant human growth hormone (hGH) in the milk of a cloned transgenic cow at levels of up to 5 g l(-1). The hormone is identical to that currently produced by expression in E. coli. In addition, the hematological and somatometric parameters of the cloned transgenic cow are within the normal range for the breed and it is fertile and capable of producing normal offspring. These results demonstrate that transgenic cattle can be used as a cost-effective alternative for the production of this hormone.

Survival of Skin Graft between Transgenic Cloned Dogs and Non-Transgenic Cloned Dogs

PubMed Central

Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Park, Jung Eun; Park, Eun Jung; Lim, Sang Hyun; Yoon, Byung Il; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2014-01-01

Whereas it has been assumed that genetically modified tissues or cells derived from somatic cell nuclear transfer (SCNT) should be accepted by a host of the same species, their immune compatibility has not been extensively explored. To identify acceptance of SCNT-derived cells or tissues, skin grafts were performed between cloned dogs that were identical except for their mitochondrial DNA (mtDNA) haplotypes and foreign gene. We showed here that differences in mtDNA haplotypes and genetic modification did not elicit immune responses in these dogs: 1) skin tissues from genetically-modified cloned dogs were successfully transplanted into genetically-modified cloned dogs with different mtDNA haplotype under three successive grafts over 63 days; and 2) non-transgenic cloned tissues were accepted into transgenic cloned syngeneic recipients with different mtDNA haplotypes and vice versa under two successive grafts over 63 days. In addition, expression of the inserted gene was maintained, being functional without eliciting graft rejection. In conclusion, these results show that transplanting genetically-modified tissues into normal, syngeneic or genetically-modified recipient dogs with different mtDNA haplotypes do not elicit skin graft rejection or affect expression of the inserted gene. Therefore, therapeutically valuable tissue derived from SCNT with genetic modification might be used safely in clinical applications for patients with diseased tissues. PMID:25372489

Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy.

PubMed

Zhao, Xueyan; Yang, Qiang; Zhao, Kewei; Jiang, Chao; Ren, Dongren; Xu, Pan; He, Xiaofang; Liao, Rongrong; Jiang, Kai; Ma, Junwu; Xiao, Shijun; Ren, Jun; Xing, Yuyun

2016-07-01

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.

Expression of recombinant human α-lactalbumin in milk of transgenic cloned pigs is sufficient to enhance intestinal growth and weight gain of suckling piglets.

PubMed

Ma, Jin; Li, Qiuyan; Li, Yan; Wen, Xiao; Li, Zhiyuan; Zhang, Zaihu; Zhang, Jiuming; Yu, Zhengquan; Li, Ning

2016-06-10

Human α-lactalbumin (HLA) has very high nutritional value and important physiological functions during the neonatal period. The peptides derived from HLA provide diverse health benefits including antimicrobial, antiviral, immune-modulating, and antihypertensive effects. Thus, it is worth investigating the effects on offspring development of increasing HLA in milk. In this study, we found that recombinant human α-lactalbumin (rHLA) exhibits efficient inhibition of dipeptidyl peptidase-IV (DPP-IV) activity in an in vitro simulated gastrointestinal digestion system. Using a BAC clone containing the complete HLA gene as a candidate vector, we generated two lines of transgenic cloned sows via somatic cell nuclear transfer that over-expressed rHLA. The average concentrations of rHLA in milk from the two lines of transgenic cloned sows were 2.24 ± 0.71 mg/ml and 2.67 ± 1.29 mg/ml. The feeding experiments revealed that rHLA represses dipeptidyl peptidase-IV (DPP-IV) activity in vivo. Furthermore, the piglets reared by rHLA transgenic cloned sows exhibit better performance in gain of body weight and intestine growth than the control piglets reared by non-transgenic sows. Therefore, these findings indicate that rHLA could serve as a natural precursor for a DPP-IV inhibitor, and the transgenic technology that produced the over-expression of rHLA could be a useful method for pig breeders to improve lactation performance. Copyright © 2016. Published by Elsevier B.V.

Handmade Cloned Transgenic Piglets Expressing the Nematode Fat-1 Gene

PubMed Central

Zhang, Peng; Zhang, Yidi; Dou, Hongwei; Yin, Jingdong; Chen, Yu; Pang, Xinzhi; Vajta, Gabor; Bolund, Lars

2012-01-01

Abstract Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been adapted worldwide, but this application is somewhat limited by its relatively low efficiency. In this study, we used handmade cloning (HMC) established previously to produce transgenic pigs that express the functional nematode fat-1 gene. Codon-optimized mfat-1 was inserted into eukaryotic expression vectors, which were transferred into primary swine donor cells. Reverse transcriptase PCR (RT-PCR), gas chromatography, and chromosome analyses were performed to select donor clones capable of converting n-6 into n-3 fatty acids. Blastocysts derived from the clones that lowered the n-6/n-3 ratio to approximately 1:1 were transferred surgically into the uteri of recipients for transgenic piglets. By HMC, 37% (n=558) of reconstructed embryos developed to the blastocyst stage after 7 days of culture in vitro, with an average cell number of 81±36 (n=14). Three recipients became pregnant after 408 day-6 blastocysts were transferred into four naturally cycling females, and a total of 14 live offspring were produced. The nematode mfat-1 effectively lowered the n-6/n-3 ratio in muscle and major organs of the transgenic pig. Our results will help to establish a reliable procedure and an efficient option in the production of transgenic animals. PMID:22686479

Production of cloned embryos from caprine mammary epithelial cells expressing recombinant human β-defensin-3.

PubMed

Liu, Jun; Luo, Yan; Liu, Qingqing; Zheng, Liming; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

2013-03-01

Transgenic animals that express antimicrobial agents in their milk can inhibit bacterial pathogens that cause mastitis. Our objective was to produce human β-defensin-3 (HBD3) transgenic embryos by nuclear transfer using goat mammary epithelial cells (GMECs) as donor cells. Three GMEC lines (GMEC1, GMEC2, and GMEC3) were transfected with a HBD3 mammary-specific expression vector by electroporation. There was a difference (P < 0.05) in the rate of geneticin-resistant colony formation among cell lines GMEC1, GMEC2, and GMEC3 (39 and 47 vs. 19 colonies per 3 × 10(6) cells, respectively). After inducing expression, the mRNA and protein of HBD3 were detected by reverse transcription polymerase chain reaction and Western blot analysis in transgenic cells. Transgenic clonal cells expressing HBD3 were used as donor cells to investigate development of cloned embryos. There were no significant differences in rates of cleavage or blastocyst formation of cloned embryos from transgenic (GMEC1T2 and GMEC2T3) and nontransgenic (GMEC1 and GMEC2) GMECs (72.3 ± 5.0%, 69.5 ± 2.3%, 61.8 ± 4.8%, and 70.0 ± 2%; and 16.8 ± 0.5%, 17.5 ± 0.7%, 16.7 ± 0.9%, and 17.5 ± 0.6%, respectively). However, the fusion rate, cleavage rate, and blastocyst formation rate of cloned embryos from a transgenic clonal cell line (GMEC2T6, 50.7 ± 2.1%, 55.5 ± 2.0%, and 11.1 ± 0.6%) were lower than those of other groups (P < 0.05). We concluded that genetic modification of GMECs might not influence the in vitro development of cloned embryos, but that some of the transgenic clonal cells were not suitable for nuclear transfer to produce transgenic goats, because of low developmental rates. However, transgenic GMECs expressing HBD3 might be used as donor cells for producing transgenic goats that express increased concentrations of β-defensins in their milk. Copyright © 2013 Elsevier Inc. All rights reserved.

Optimization of embryo culture conditions for increasing efficiency of cloning in buffalo (Bubalus bubalis) and generation of transgenic embryos via cloning.

PubMed

Wadhwa, Neerja; Kunj, Neetu; Tiwari, Shuchita; Saraiya, Megha; Majumdar, Subeer S

2009-09-01

Cloning in bovine species is marred by low efficiency of blastocyst formation. Any increase in the efficiency of blastocyst formation upon nuclear transfer will greatly enhance the efficiency of cloning. In the present study, the effect of various media, protein sources, and growth factors on the development of cloned buffalo embryos was evaluated. Among various combinations tested, culture of cloned embryos in TCM-199 media on the feeder layer of Buffalo Oviductal Epithelial Cells (BOEC) in the presence of bovine serum albumin-free fatty acid (BSA-FFA) and leukemia inhibitory factor (LIF) provided most suitable environment for efficient development of cloned blastocysts. Under these conditions, we achieved a blastocyst formation rate of 43%, which is better than those reported previously. Because preimplantation embryonic development, in vivo, occurs in an environment of oviductal cells, the blastocysts generated by this method may presumably be more suitable for implantation and further development. Additionally, we generated green blastocysts from enucleated oocytes by transfer of nuclei from cells transfected with EGFP transgene, showing possibility of transgenesis via cloning in this species. To our knowledge, this is the first report regarding the production of transgenic cloned buffalo embryos and their developmental competence with respect to various media, cocultures, and supplements.

A 90-day safety study in Sprague-Dawley rats fed milk powder containing recombinant human lactoferrin (rhLF) derived from transgenic cloned cattle.

PubMed

Zhou, Cui; Wang, Jian Wu; Huang, Kun Lun; He, XiaoYun; Chen, Xiu Ping; Sun, Hong; Yu, Tian; Che, Hui Lian

2011-10-01

Transgenic cloned animals expressing beneficial human nutritional traits offer a new strategy for large-scale production of some kinds of functional substances. In some cases, the required safety testing for genetically modified (GM) foods do not seem appropriate for human food safety, though regulations do not seem to provide alternatives. A 90-day rat feeding study is the core study for the safety assessment of GM foods. The test material in this 90-day study was prepared nonfat milk powder containing recombinant human lactoferrin (rhLF), which was expressed in transgenic cloned cattle. Groups of 10 male and female Sprague-Dawley rats were given a nutritionally balanced purified diet containing 7.5, 15, or 30% transgenic or conventional milk powder for 90 days. A commercial AIN93G diet was used as an additional control group. Clinical, biological, and pathological parameters were compared between groups. The only significant effect of treatment was higher mean ferritin and Fe(+) concentrations for both male and female rats fed the transgenic milk powder diets, as compared to rats fed nontransgenic milk diets or the commercial diet. The results of the present study are consistent with previous research, which indicates that milk powder containing rhLF derived from healthy transgenic cloned cattle is as safe as conventional milk powder.

Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange.

PubMed

Matsunari, Hitomi; Onodera, Masafumi; Tada, Norihiro; Mochizuki, Hideki; Karasawa, Satoshi; Haruyama, Erika; Nakayama, Naoki; Saito, Hitoshi; Ueno, Satoshi; Kurome, Mayuko; Miyawaki, Atsushi; Nagashima, Hiroshi

2008-09-01

Genetically engineered pigs with cell markers such as fluorescent proteins are highly useful in lines of research that include the tracking of transplanted cells or tissues. In this study, we produced transgenic-cloned pigs carrying a gene for the newly developed red fluorescent protein, humanized Kusabira-Orange (huKO), which was cloned from the coral stone Fungia concinna. The nuclear transfer embryos, reconstructed with fetal fibroblast cells that had been transduced with huKO cDNA using retroviral vector D Delta Nsap, developed efficiently in vitro into blastocysts (28.0%, 37/132). Nearly all (94.6%, 35/37) of the cloned blastocysts derived from the transduced cells exhibited clear huKO gene expression. A total of 429 nuclear transfer embryos were transferred to four recipients, all of which became pregnant and gave birth to 18 transgenic-cloned offspring in total. All of the pigs highly expressed huKO fluorescence in all of the 23 organs and tissues analyzed, including the brain, eyes, intestinal and reproductive organs, skeletal muscle, bone, skin, and hoof. Furthermore, such expression was also confirmed by histological analyses of various tissues such as pancreatic islets, renal corpuscles, neuronal and glial cells, the retina, chondrocytes, and hematopoietic cells. These data demonstrate that transgenic-cloned pigs exhibiting systemic red fluorescence expression can be efficiently produced by nuclear transfer of somatic cells retrovirally transduced with huKO gene.

Large-scale production of functional human lysozyme from marker-free transgenic cloned cows.

PubMed

Lu, Dan; Liu, Shen; Ding, Fangrong; Wang, Haiping; Li, Jing; Li, Ling; Dai, Yunping; Li, Ning

2016-03-10

Human lysozyme is an important natural non-specific immune protein that is highly expressed in breast milk and participates in the immune response of infants against bacterial and viral infections. Considering the medicinal value and market demand for human lysozyme, an animal model for large-scale production of recombinant human lysozyme (rhLZ) is needed. In this study, we generated transgenic cloned cows with the marker-free vector pBAC-hLF-hLZ, which was shown to efficiently express rhLZ in cow milk. Seven transgenic cloned cows, identified by polymerase chain reaction, Southern blot, and western blot analyses, produced rhLZ in milk at concentrations of up to 3149.19 ± 24.80 mg/L. The purified rhLZ had a similar molecular weight and enzymatic activity as wild-type human lysozyme possessed the same C-terminal and N-terminal amino acid sequences. The preliminary results from the milk yield and milk compositions from a naturally lactating transgenic cloned cow 0906 were also tested. These results provide a solid foundation for the large-scale production of rhLZ in the future.

Somatic cell nuclear transfer cloning: practical applications and current legislation.

PubMed

Niemann, H; Lucas-Hahn, A

2012-08-01

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved. © 2012 Blackwell Verlag GmbH.

Ars insulator identified in sea urchin possesses an activity to ensure the transgene expression in mouse cells.

PubMed

Tajima, Shoji; Shinohara, Keiko; Fukumoto, Maiko; Zaitsu, Reiko; Miyagawa, Junichi; Hino, Shinjiro; Fan, Jun; Akasaka, Koji; Matsuoka, Masao

2006-04-01

Sea urchin arylsulfatase (Ars) gene locus has features of an insulator, i.e., blocking of enhancer and promoter interaction, and protection of a transgene against positional effects [Akasaka et al. (1999) Cell. Mol. Biol. 45, 555-565]. To examine the effect of Ars insulator on long-term expression of a transgene, the insulator was inserted into LTR of retrovirus vector harboring hrGFP gene as a reporter, and then introduced into mouse myoblast cells. The isolated clones transduced with the reporter gene with or without Ars insulator were cultured for more than 20 wk in the absence of a selection reagent, and the expression of hrGFP was periodically determined. Expression of hrGFP in four clones transduced with the reporter gene without Ars insulator was completely silenced after 20 wk of culture. On the other hand, hrGFP was expressed in all clones with Ars insulator inserted in one of the two different orientations. Histone H3 deacetylation and DNA methylation of the 5'LTR promoter region, signs for heterochromatin and silencing, were suppressed in the clones that were expressing hrGFP. Ars insulator is effective in maintaining a transgene in mouse cells in an orientation-dependent manner, and will be a useful tool to ensure stable expression of a transgene.

The transgenic cloned pig population with integrated and controllable GH expression that has higher feed efficiency and meat production

PubMed Central

Ju, Huiming; Zhang, Jiaqing; Bai, Lijing; Mu, Yulian; Du, Yutao; Yang, Wenxian; Li, Yong; Sheng, Anzhi; Li, Kui

2015-01-01

Sustained expression of the GH gene has been shown to have detrimental effects on the health of animals. In the current study, transgenic founder pigs, with controllable pig growth hormone (pGH) expression, were cloned via the handmade cloning method (HMC), and pGH expression levels were examined at the cellular and organismal levels. The serum pGH levels in 3 founder male pigs were found to be significantly higher after induction with intramuscular injection of doxycycline (DOX) compared to baseline. A daily dose of DOX was administered via feed to these animals for a period of 65 to 155 days. The growth rate, feed efficiency and pGH serum concentration increased in the DOX-induced transgenic group compared with the other groups. 8 numbers of animals were euthanized and the dressing percentage, loin muscle and lean meat percentage were significantly higher in the DOX-induced F1 transgenic group compared with the other groups. In this study a large population of transgenic pigs, with integrated controllable expression of a transgene, was obtained. The transgenic pigs were healthy and normal in terms of reproductive capability. At the same time, feed efficiency was improved, production processes were accelerated and meat yield was increased. PMID:25959098

The transgenic cloned pig population with integrated and controllable GH expression that has higher feed efficiency and meat production.

PubMed

Ju, Huiming; Zhang, Jiaqing; Bai, Lijing; Mu, Yulian; Du, Yutao; Yang, Wenxian; Li, Yong; Sheng, Anzhi; Li, Kui

2015-05-11

Sustained expression of the GH gene has been shown to have detrimental effects on the health of animals. In the current study, transgenic founder pigs, with controllable pig growth hormone (pGH) expression, were cloned via the handmade cloning method (HMC), and pGH expression levels were examined at the cellular and organismal levels. The serum pGH levels in 3 founder male pigs were found to be significantly higher after induction with intramuscular injection of doxycycline (DOX) compared to baseline. A daily dose of DOX was administered via feed to these animals for a period of 65 to 155 days. The growth rate, feed efficiency and pGH serum concentration increased in the DOX-induced transgenic group compared with the other groups. 8 numbers of animals were euthanized and the dressing percentage, loin muscle and lean meat percentage were significantly higher in the DOX-induced F1 transgenic group compared with the other groups. In this study a large population of transgenic pigs, with integrated controllable expression of a transgene, was obtained. The transgenic pigs were healthy and normal in terms of reproductive capability. At the same time, feed efficiency was improved, production processes were accelerated and meat yield was increased.

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

PubMed

Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

2015-01-01

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

Bone marrow mesenchymal stem cells are an attractive donor cell type for production of cloned pigs as well as genetically modified cloned pigs by somatic cell nuclear transfer.

PubMed

Li, Zicong; He, Xiaoyan; Chen, Liwen; Shi, Junsong; Zhou, Rong; Xu, Weihua; Liu, Dewu; Wu, Zhenfang

2013-10-01

The somatic cell nuclear transfer (SCNT) technique has been widely applied to clone pigs or to produce genetically modified pigs. Currently, this technique relies mainly on using terminally differentiated fibroblasts as donor cells. To improve cloning efficiency, only partially differentiated multipotent mesenchymal stem cells (MSCs), thought to be more easily reprogrammed to a pluripotent state, have been used as nuclear donors in pig SCNT. Although in vitro-cultured embryos cloned from porcine MSCs (MSCs-embryos) were shown to have higher preimplantation developmental ability than cloned embryos reconstructed from fibroblasts (Fs-embryos), the difference in in vivo full-term developmental rate between porcine MSCs-embryos and Fs-embryos has not been investigated so far. In this study, we demonstrated that blastocyst total cell number and full-term survival abilities of MSCs-embryos were significantly higher than those of Fs-embryos cloned from the same donor pig. The enhanced developmental potential of MSCs-embryos may be associated with their nuclear donors' DNA methylation profile, because we found that the methylation level of imprinting genes and repeat sequences differed between MSCs and fibroblasts. In addition, we showed that use of transgenic porcine MSCs generated from transgene plasmid transfection as donor cells for SCNT can produce live transgenic cloned pigs. These results strongly suggest that porcine bone marrow MSCs are a desirable donor cell type for production of cloned pigs and genetically modified cloned pigs via SCNT.

Regulation of animal biotechnology: research needs.

PubMed

Rexroad, C E; Green, R D; Wall, R J

2007-09-01

Livestock that result from biotechnology have been a part of agricultural science for over 30 years but have not entered the market place as food or fiber. Two biotechnologies are at the forefront as challenges to the world's systems for regulating the market place: animal clones and transgenic animals. Both technologies have come before the Food and Drug Administration in the United States and it appears that action is imminent for clones. The FDA has asserted principles for evaluation of clones and asserts that "... remaining hazard(s) from cloning are likely to be subtle in nature." The science-based principles recognize that in some areas related to developmental biology and gene expression in clones, additional scientific information would be useful. The role of science then is to use the genomic tools that we have available to answer questions about epigenetic regulation of development and reprogramming of genes to the state found in germ cells. Transgenics pose additional challenges to regulators. If the transgenics are produced using cloning from modified cells then the additional scientific information needed will be related to the effects of insertion and expression of the transgenes. Other approaches such as retrovirally vectored transgenesis will elicit additional questions. These questions will be challenging because the science will have to be related to the expression and function of each gene or class of genes. For the promises of animal biotechnology to be fulfilled, scientists will have to resolve many questions for regulators and the public but tools to answer those questions are rapidly becoming available.

Cloning: Past, Present, and the Exciting Future. Breakthroughs in Bioscience.

ERIC Educational Resources Information Center

Di Berardino, Marie A.

This document explores the history of cloning by focusing on Dolly the Sheep, one of the first large animal clonings. The disadvantages and advantages of transgenic clones are discussed as well as the future implications of cloning from the perspective of human health. (Contains 10 resources.) (YDS)

Screening somatic cell nuclear transfer parameters for generation of transgenic cloned cattle with intragenomic integration of additional gene copies that encode bovine adipocyte-type fatty acid-binding protein (A-FABP).

PubMed

Guo, Yong; Li, Hejuan; Wang, Ying; Yan, Xingrong; Sheng, Xihui; Chang, Di; Qi, Xiaolong; Wang, Xiangguo; Liu, Yunhai; Li, Junya; Ni, Hemin

2017-02-01

Somatic cell nuclear transfer (SCNT) is frequently used to produce transgenic cloned livestock, but it is still associated with low success rates. To our knowledge, we are the first to report successful production of transgenic cattle that overexpress bovine adipocyte-type fatty acid binding proteins (A-FABPs) with the aid of SCNT. Intragenomic integration of additional A-FABP gene copies has been found to be positively correlated with the intramuscular fat content in different farm livestock species. First, we optimized the cloning parameters to produce bovine embryos integrated with A-FABP by SCNT, such as applied voltage field strength and pulse duration for electrofusion, morphology and size of donor cells, and number of donor cells passages. Then, bovine fibroblast cells from Qinchuan cattle were transfected with A-FABP and used as donor cells for SCNT. Hybrids of Simmental and Luxi local cattle were selected as the recipient females for A-FABP transgenic SCNT-derived embryos. The results showed that a field strength of 2.5 kV/cm with two 10-μs duration electrical pulses was ideal for electrofusion, and 4-6th generation circular smooth type donor cells with diameters of 15-25 μm were optimal for producing transgenic bovine embryos by SCNT, and resulted in higher fusion (80%), cleavage (73%), and blastocyst (27%) rates. In addition, we obtained two transgenic cloned calves that expressed additional bovine A-FABP gene copies, as detected by PCR-amplified cDNA sequencing. We proposed a set of optimal protocols to produce transgenic SCNT-derived cattle with intragenomic integration of ectopic A-FABP-inherited exon sequences.

Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca

USDA-ARS?s Scientific Manuscript database

Transgenic clones C2, C3, C4, C5, C6, PT3 and PT5 of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were evaluated for Sharka resistance under high infection pressure in field trials in Romania. Transgenic clone C5, subsequently named "HoneySweet", showed high re...

IgE reactivity to profilin in pollen-sensitized subjects with adverse reactions to banana and pineapple.

PubMed

Reindl, J; Rihs, H P; Scheurer, S; Wangorsch, A; Haustein, D; Vieths, S

2002-06-01

The so-called 'latex-fruit syndrome' is a well-documented phenomenon in cross-reactive allergies. By contrast, there is a lack of information about allergy to exotic fruits in patients with a predominant pollen sensitization. Since the ubiquitous protein profilin has been identified as an allergen in natural rubber latex as well as in pollen-related foods, the aim of this study was to investigate the role of profilin in allergy to certain exotic fruits. Recombinant profilins from banana and pineapple were cloned by a PCR technique after isolation of total RNA using degenerated profilin-specific primers. The unknown 5' ends of copy DNA (cDNA) were identified by rapid amplification of 5'cDNA ends (5'-RACE) and expression in Escherichia coli BL21(DE3) cells. The recombinant profilins were purified by affinity chromatography using poly-(L)-proline as the solid phase. IgE-binding capabilities were characterized by means of immunoblot and Enzyme Allergosorbent Test (EAST). The cross-reactivity to birch pollen profilin and latex profilin was studied by EAST as well as by immunoblot inhibition experiments. Both banana and pineapple profilin were found to consist of 131 amino acid residues with high amino acid sequence identity to known allergenic pollen and food profilins (71-84%). IgE binding to the recombinant profilins was observed in 7/16 sera from subjects with suspected banana allergy (44%) and in 8/19 sera from subjects with suspected pineapple allergy (42%). Inhibition experiments indicated similar IgE reactivity of natural and recombinant allergens. In addition, high cross-reactivity to birch pollen profilin Bet v 2 and latex profilin Hev b 8 was demonstrated by immunoblot inhibition as well as EAST inhibition experiments. Since a high IgE-binding prevalence of about 40% was obtained in both banana and pineapple allergy, we conclude that profilin is an important mediator of IgE cross-reactivity between pollen and exotic fruits. Copyright 2002 S. Karger AG, Basel

Bone Marrow Mesenchymal Stem Cells Are an Attractive Donor Cell Type for Production of Cloned Pigs As Well As Genetically Modified Cloned Pigs by Somatic Cell Nuclear Transfer

PubMed Central

Li, Zicong; He, Xiaoyan; Chen, Liwen; Shi, Junsong; Zhou, Rong; Xu, Weihua

2013-01-01

Abstract The somatic cell nuclear transfer (SCNT) technique has been widely applied to clone pigs or to produce genetically modified pigs. Currently, this technique relies mainly on using terminally differentiated fibroblasts as donor cells. To improve cloning efficiency, only partially differentiated multipotent mesenchymal stem cells (MSCs), thought to be more easily reprogrammed to a pluripotent state, have been used as nuclear donors in pig SCNT. Although in vitro–cultured embryos cloned from porcine MSCs (MSCs-embryos) were shown to have higher preimplantation developmental ability than cloned embryos reconstructed from fibroblasts (Fs-embryos), the difference in in vivo full-term developmental rate between porcine MSCs-embryos and Fs-embryos has not been investigated so far. In this study, we demonstrated that blastocyst total cell number and full-term survival abilities of MSCs-embryos were significantly higher than those of Fs-embryos cloned from the same donor pig. The enhanced developmental potential of MSCs-embryos may be associated with their nuclear donors' DNA methylation profile, because we found that the methylation level of imprinting genes and repeat sequences differed between MSCs and fibroblasts. In addition, we showed that use of transgenic porcine MSCs generated from transgene plasmid transfection as donor cells for SCNT can produce live transgenic cloned pigs. These results strongly suggest that porcine bone marrow MSCs are a desirable donor cell type for production of cloned pigs and genetically modified cloned pigs via SCNT. PMID:24033142

Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin.

PubMed

Wan, Yong-Jie; Zhang, Yan-Li; Zhou, Zheng-Rong; Jia, Ruo-Xin; Li, Meng; Song, Hui; Wang, Zi-Yu; Wang, Li-Zhong; Zhang, Guo-Min; You, Ji-Hao; Wang, Feng

2012-08-01

The objective was to investigate the effects of the transgenic donor cell synchronization method, oocyte sources, and other factors, on production of hLF-gene nucleus transfer dairy goats. Three transfected cell lines from ear biopsies from three 3-mo-old Saanen dairy goats (designated Number 1, Number 2, and Number 3, respectively) were selected as karyoplast donors for somatic cell nuclear transfer (SCNT) after detailed identification (including PCR and sequencing of PCR products). In donor cell cycle synchronization studies, the apoptosis rate of hLF transgenic fibroblasts was not different (P > 0.05) after 3 days of serum starvation or 2 days of contact inhibition. Additionally, there was no effect (P > 0.05) on developmental capacity of reconstructed embryos; however, the kidding rate of recipients in the serum starvation group was higher than that in the contact inhibition group (18 vs. 0%, respectively). The production efficiency of the transgenic cloned goats using donor cells from the Number 1 dairy goat cell line was higher than those using the Number 2 and the Number 3 cell lines (kidding rates were 18, 2, and 0%, respectively, P < 0.05). The oocyte source did not significantly affect the pregnancy rate of hLF-transgenic cloned dairy goats, but more fetuses were aborted when using in vitro matured oocytes compared to in vivo matured oocytes. In summary, utilizing transfected 3-mo-old dairy goat fibroblasts as donor cells, seven live offspring were produced, and the hLF gene was successfully integrated. This study provided additional insights into preparation of donor cells and recipient oocytes for producing transgenic cloned goats through SCNT. Copyright © 2012 Elsevier Inc. All rights reserved.

An efficient transgenic system by TA cloning vectors and RNAi for C. elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gengyo-Ando, Keiko; CREST, JST, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012; Yoshina, Sawako

2006-11-03

In the nematode, transgenic analyses have been performed by microinjection of DNA from various sources into the syncytium gonad. To expedite these transgenic analyses, we solved two potential problems in this work. First, we constructed an efficient TA-cloning vector system which is useful for any promoter. By amplifying the genomic DNA fragments which contain regulatory sequences with or without the coding region, we could easily construct plasmids expressing fluorescent protein fusion without considering restriction sites. We could dissect motor neurons with three colors in a single animal. Second, we used feeding RNAi to isolate transgenic strains which express lag-2::venus fusionmore » gene. We found that the fusion protein is toxic when ectopically expressed in embryos but is functional to rescue a loss of function mutant in the lag-2 gene. Thus, the transgenic system described here should be useful to examine the protein function in the nematode.« less

Use of Chenopodium murale L. transgenic hairy root in vitro culture system as a new tool for allelopathic assays.

PubMed

Mitić, Nevena; Dmitrović, Slavica; Djordjević, Mirka; Zdravković-Korać, Snežana; Nikolić, Radomirka; Raspor, Martin; Djordjević, Tatjana; Maksimović, Vuk; Zivković, Suzana; Krstić-Milošević, Dijana; Stanišić, Mariana; Ninković, Slavica

2012-08-15

We investigated Chenopodium murale transgenic hairy root in vitro culture system as a new tool for allelopathic assays. Transgenic hairy roots were induced by Agrobacterium rhizogenes A4M70GUS from roots, cotyledons, leaves, and internodes of C. murale seedlings. Roots were found to be the best target explants, providing transformation efficiency of up to 11.1%. Established hairy root clones differed in their morphology and growth potential. Molecular characterization of these clones was carried out by PCR, RT-PCR and histochemical GUS analyses. No differences in rol gene expression were observed. Liquid culture system of characterized hairy root clones was maintained for over 2 years. Six hairy root clones were selected for assaying the allelopathic effect of their growth medium against germination and seedling elongation of wheat and lettuce test plants. The inhibitory potential varied depending on the hairy root clone. Some transgenic clones showed significantly higher inhibition compared to wild-type roots. These results revealed that hairy roots as an independent system synthesize some bioactive substances with allelopathic activity and exude them into the growth medium. Concentrations of caffeic, ferulic and p-coumaric acids (0.07-2.85 μmol/L) identified by HPLC analysis in the growth media were at least 1000 times lower than the inhibitory active concentration (5 mmol/L) of pure grade phenolic acids, suggesting that they have a limited role in the allelopathic phenomena of C. murale. The presented hairy root system appears to be a suitable tool for further investigation of the potential and nature of root-mediated allelopathic interference of C. murale. Copyright © 2012 Elsevier GmbH. All rights reserved.

Transgenic and cloned animals in the food chain--are we prepared to tackle it?

PubMed

Jagadeesan, Premanandh; Bin Salem, Samara

2015-11-01

Transgenic and cloned animal production for various purposes has been increasing rapidly in recent times. While the actual impact of these animals in the food chain is unknown, the significance of tracking and monitoring measures to curb accidental and or deliberate release has been discussed. Religious perspectives from different faiths and traditions have been presented. Although the concept of substantial equivalence satisfies the technical and nutritional requirements of these products when assessed against comparators, public opinion and religious concerns should also be considered by the regulators while developing policy regulations. In conclusion, measures to prevent real or perceived risks of transgenic and cloned animals in food production require global coordinated action. It is worthwhile to consider establishing effective tracking systems and analytical procedures as this will be a valuable tool if a global consensus is not reached on policy regulation. © 2015 Society of Chemical Industry.

Establishment of a canine model of human type 2 diabetes mellitus by overexpressing phosphoenolypyruvate carboxykinase.

PubMed

Jeong, Yeon Woo; Lee, Geun-Shik; Kim, Joung Joo; Park, Sun Woo; Ko, Kyeong Hee; Kang, Mina; Kim, Yu Kyung; Jung, Eui-Man; Hyun, Sang Hwan; Shin, Taeyoung; Jeung, Eui-Bae; Hwang, Woo Suk

2012-08-01

Dogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transfer (SCNT) facilitates the production of transgenic dogs. In this study, we generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus. In addition, we assessed the cloning efficiency associated with adult or fetal (cloned or natural mating) fibroblasts as a nuclear source. Cloning efficiency was determined by the fusion, pregnancy and cloning rates. The fusion rates were significantly high for fibroblasts from cloned fetuses, but the pregnancy and cloning rates were relatively high for cells from normal fetuses. Based on these data, fetal fibroblasts were selected as the nuclear donor for SCNT and genetically engineered to overexpress the PEPCK gene and dual selection marker genes controlled by the PEPCK promoter. The transgenic cells were introduced into oocytes and transferred into five recipient dogs, resulting in two pregnancies. Finally, three puppies were born and confirmed by microsatellite analysis to be genetically identical to the donor. One puppy successfully overexpressed PEPCK mRNA and protein in the liver. This canine disease model may be useful for studying the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus.

Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer.

PubMed

Feng, Xiujing; Cao, Shaoxian; Wang, Huili; Meng, Chunhua; Li, Jingxin; Jiang, Jin; Qian, Yong; Su, Lei; He, Qiang; Zhang, Qingxiao

2015-02-01

Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat β-lactoglobulin (βLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

[Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers].

PubMed

An, Liyou; Yuan, Yuguo; Yu, Baoli; Yang, Tingjia; Cheng, Yong

2012-12-01

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

Phenolic Compounds and Expression of 4CL Genes in Silver Birch Clones and Pt4CL1a Lines

PubMed Central

Sutela, Suvi; Hahl, Terhi; Tiimonen, Heidi; Aronen, Tuija; Ylioja, Tiina; Laakso, Tapio; Saranpää, Pekka; Chiang, Vincent; Julkunen-Tiitto, Riitta; Häggman, Hely

2014-01-01

A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins. PMID:25502441

Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of transgenic cloned cows.

PubMed

Wang, Yuhang; Ding, Fangrong; Wang, Tao; Liu, Wenjie; Lindquist, Susanne; Hernell, Olle; Wang, Jianwu; Li, Jing; Li, Ling; Zhao, Yaofeng; Dai, Yunping; Li, Ning

2017-01-01

Bile salt-stimulated lipase (BSSL) is a lipolytic digestive enzyme with broad substrate specificity secreted from exocrine pancreas into the intestinal lumen in all species and from the lactating mammary gland into the milk of some species, notably humans but not cows. BSSL in breast milk facilitates digestion and absorption of milk fat and promotes growth of small for gestational age preterm infants. Thus, purified recombinant human BSSL (rhBSSL) can be used for treatment of patients with fat malabsorption and expressing rhBSSL in the milk of transgenic cloned cows would therefore be a mean to meet a medical need. In the present study, a vector pBAC-hLF-hBSSL was constructed, which efficiently expressed active rhBSSL in milk of transgenic cloned cows to a concentration of 9.8 mg/ml. The rhBSSL purified from cow milk had the same enzymatic activity, N-terminal amino acid sequence, amino acid composition and isoelectric point and similar physicochemical characteristics as human native BSSL. Our study supports the use of transgenic cattle for the cost-competitive, large-scale production of therapeutic rhBSSL.

Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of transgenic cloned cows

PubMed Central

Ding, Fangrong; Wang, Tao; Liu, Wenjie; Lindquist, Susanne; Hernell, Olle; Wang, Jianwu; Li, Jing; Li, Ling; Zhao, Yaofeng; Dai, Yunping; Li, Ning

2017-01-01

Bile salt-stimulated lipase (BSSL) is a lipolytic digestive enzyme with broad substrate specificity secreted from exocrine pancreas into the intestinal lumen in all species and from the lactating mammary gland into the milk of some species, notably humans but not cows. BSSL in breast milk facilitates digestion and absorption of milk fat and promotes growth of small for gestational age preterm infants. Thus, purified recombinant human BSSL (rhBSSL) can be used for treatment of patients with fat malabsorption and expressing rhBSSL in the milk of transgenic cloned cows would therefore be a mean to meet a medical need. In the present study, a vector pBAC-hLF-hBSSL was constructed, which efficiently expressed active rhBSSL in milk of transgenic cloned cows to a concentration of 9.8 mg/ml. The rhBSSL purified from cow milk had the same enzymatic activity, N-terminal amino acid sequence, amino acid composition and isoelectric point and similar physicochemical characteristics as human native BSSL. Our study supports the use of transgenic cattle for the cost-competitive, large-scale production of therapeutic rhBSSL. PMID:28475629

Production and Breeding of Transgenic Cloned Pigs Expressing Human CD73.

PubMed

Lee, Seung-Chan; Lee, Haesun; Oh, Keon Bong; Hwang, In-Sul; Yang, Hyeon; Park, Mi-Ryung; Ock, Sun-A; Woo, Jae-Seok; Im, Gi-Sun; Hwang, Seongsoo

2017-06-01

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were 127 ± 18.9. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as α-1,3-galactosyltransferase knock-out /hCD46 for xenotransplantation.

Transgenic mammalian species, generated by somatic cell cloning, in biomedicine, biopharmaceutical industry and human nutrition/dietetics--recent achievements.

PubMed

Samiec, M; Skrzyszowska, M

2011-01-01

Somatic cell cloning technology in mammals promotes the multiplication of productively-valuable genetically engineered individuals, and consequently allows also for standardization of transgenic farm animal-derived products, which, in the context of market requirements, will have growing significance. Gene farming is one of the most promising areas in modern biotechnology. The use of live bioreactors for the expression of human genes in the lactating mammary gland of transgenic animals seems to be the most cost-effective method for the production/processing of valuable recombinant therapeutic proteins. Among the transgenic farm livestock species used so far, cattle, goats, sheep, pigs and rabbits are useful candidates for the expression of tens to hundreds of grams of genetically-engineered proteins or xenogeneic biopreparations in the milk. At the beginning of the new millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on recombinant human proteins. The ever-growing demand for such pharmaceutical or nutriceutical proteins is an important driving force for the development of safe and large-scale production platforms. The aim of this paper is to present an overall survey of the state of the art in investigations which provide the current knowledge for deciphering the possibilities of practical application of the transgenic mammalian species generated by somatic cell cloning in biomedicine, the biopharmaceutical industry, human nutrition/dietetics and agriculture.

Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human β1 and α2 Integrin

PubMed Central

Staunstrup, Nicklas Heine; Madsen, Johannes; Primo, Maria Nascimento; Li, Juan; Liu, Ying; Kragh, Peter M.; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Svensson, Lars; Petersen, Thomas K.; Callesen, Henrik; Bolund, Lars; Mikkelsen, Jacob Giehm

2012-01-01

Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis. PMID:22590584

Overview on the investigations of transgenic plums in Romania

USDA-ARS?s Scientific Manuscript database

Transgenic plums of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were the subjects of three experiments undertaken in Romania. In the first experiment, PPV-CP transgenic clones C2, C3, C4, C5, C6, PT3 and PT5 were evaluated for Sharka resistance under high natu...

Overview of the investigation of transgenic plums in Romania

USDA-ARS?s Scientific Manuscript database

Transgenic plums of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were the subjects of three experiments undertaken in Romania. In the first experiment, PPV-CP transgenic clones C2, C3, C4, C5, C6 and PT3 were evaluated for Sharka resistance under high natural i...

Micropropagation of transgenic lettuce containing HBsAg as a method of mass-scale production of standardised plant material for biofarming purposes.

PubMed

Pniewski, Tomasz; Czyż, Marcin; Wyrwa, Katarzyna; Bociąg, Piotr; Krajewski, Paweł; Kapusta, Józef

2017-01-01

Micropropagation protocol of transgenic lettuce bearing S-, M- and L-HBsAg was developed for increased production of uniformised material for oral vaccine preparation. Effective manufacturing of plant-based biopharmaceuticals, including oral vaccines, depends on sufficient content of a protein of interest in the initial material and its efficient conversion into an administrable formulation. However, stable production of plants with a uniformised antigen content is equally important for reproducible processing. This can be provided by micropropagation techniques. Here, we present a protocol for micropropagation of transgenic lettuce lines bearing HBV surface antigens: S-, M- and L-HBsAg. These were multiplied through axillary buds to avoid the risk of somaclonal variation. Micropropagation effectiveness reached 3.5-5.7 per passage, which implies potential production of up to 6600 plant clones within a maximum 5 months. Multiplication and rooting rates were statistically homogenous for most transgenic and control plants. For most lines, more than 90 % of clones obtained via in vitro micropropagation had HBsAg content as high as reference plants directly developed from seeds. Clones were also several times more uniform in HBsAg expression. Variation coefficients of HBsAg content did not exceed 10 % for approximately 40-85 % of clones, or reached a maximum 20 % for 90 % of all clones. Tissue culture did not affect total and leaf biomass yields. Seed production for clones was decreased insignificantly and did not impact progeny condition. Micropropagation facilitates a substantial increase in the production of lettuce plants with high and considerably equalised HBsAg contents. This, together with the previously reported optimisation of plant tissue processing and its long-term stability, constitutes a successive step in manufacturing of a standardised anti-HBV oral vaccine of reliable efficacy.

Optimisation of microwave-assisted processing in production of pineapple jam

NASA Astrophysics Data System (ADS)

Ismail, Nur Aisyah Mohd; Abdullah, Norazlin; Muhammad, Norhayati

2017-10-01

Pineapples are available all year round since they are unseasonal fruits. Due to the continuous harvesting of the fruit, the retailers and farmers had to find a solution such as the processing of pineapple into jam, to treat the unsuccessfully sold pineapples. The direct heating of pineapple puree during the production of pineapple jam can cause over degradation of quality of the fresh pineapple. Thus, this study aims to optimise the microwave-assisted processing conditions for producing pineapple jam which could reduce water activity and meets minimum requirement for pH and total soluble solids contents of fruit jam. The power and time of the microwave processing were chosen as the factors, while the water activity, pH and total soluble solids (TSS) content of the pineapple jam were determined as responses to be optimised. The microwave treatment on the pineapple jam was able to give significant effect on the water activity and TSS content of the pineapple jam. The optimum power and time for the microwave processing of pineapple jam is 800 Watt and 8 minutes, respectively. The use of domestic microwave oven for the pineapple jam production results in acceptable pineapple jam same as conventional fruit jam sold in the marketplace.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

PubMed

Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G; Martin, Francisco

2016-11-17

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells

PubMed Central

Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G.; Martin, Francisco

2016-01-01

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny. PMID:27853296

Chromatin insulation by a transcriptional activator

PubMed Central

Sutter, Nathan B.; Scalzo, David; Fiering, Steven; Groudine, Mark; Martin, David I. K.

2003-01-01

In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression. PMID:12547916

Confirmation of germ-line transmission in the red fluorescence protein (RFP) transgenic cloned male cat.

PubMed

Cho, Su-Jin; Lee, Young S; Lee, Jae-Ik; Bang, Jae-Il; Yang, Jing; Klassen, Henry; Kong, Il-Keun

2010-12-01

The production of transgenic animals is highly desirable for biotechnology and basic research. We investigated the reproductive ability of a red fluorescence protein (RFP) transgenic cloned male cat (RFP TG cat) by natural mating with a domestic female cat. The RFP expression levels were examined in early embryogenesis, tissues from 45-day-old two fetuses, and four RFP TG cat offspring. The RFP gene was detected in tissue samples from one dead kitten, including several organs and the skin. Also, under a fluorescent light source, we were able to directly detect the RFP expression of in in vitro-produced blastocysts derived with sperm from the RFP TG cat. These results indicate that the RFP TG cat exhibits normal reproductive fertility, stable germ-line transmission of the RFP transgene, and characteristic RFP expression in its offspring. We isolated feline neural progenitor cells from a 45-day-old fetus derived from the natural mating of the RFP TG cat with a domestic female cat. Isolated brain and retinal progenitor cells were successfully passaged at least four times post isolation (day 23), and showed a high RFP expression level. This method of producing genetically modified cloned cats will be important for generating biomedical models of human diseases.

Silencing the HaHR3 Gene by Transgenic Plant-mediated RNAi to Disrupt Helicoverpa armigera Development

PubMed Central

Xiong, Yehui; Zeng, Hongmei; Zhang, Yuliang; Xu, Dawei; Qiu, Dewen

2013-01-01

RNA interference (RNAi) caused by exogenous double-stranded RNA (dsRNA) has developed into a powerful technique in functional genomics, and to date it is widely used to down-regulate crucial physiology-related genes to control pest insects. A molt-regulating transcription factor gene, HaHR3, of cotton bollworm (Helicoverpa armigera) was selected as the target gene. Four different fragments covering the coding sequence (CDS) of HaHR3 were cloned into vector L4440 to express dsRNAs in Escherichia coli. The most effective silencing fragment was then cloned into a plant over-expression vector to express a hairpin RNA (hpRNA) in transgenic tobacco (Nicotiana tabacum). When H. armigera larvae were fed the E. coli or transgenic plants, the HaHR3 mRNA and protein levels dramatically decreased, resulting developmental deformity and larval lethality. The results demonstrate that both recombinant bacteria and transgenic plants could induce HaHR3 silence to disrupt H. armigera development, transgenic plant-mediated RNAi is emerging as a powerful approach for controlling insect pests. PMID:23630449

Flower induction, microscope-aided cross-pollination, and seed production in the duckweed Lemna gibba with discovery of a male-sterile clone.

PubMed

Fu, Lili; Huang, Meng; Han, Bingying; Sun, Xuepiao; Sree, K Sowjanya; Appenroth, Klaus-J; Zhang, Jiaming

2017-06-08

Duckweed species have a great potential to develop into fast-growing crops for water remediation and bioenergy production. Seed production and utilization of hybrid vigour are essential steps in this process. However, even in the extensively-studied duckweed species, Lemna gibba, flower primordia were often aborted prior to maturation. Salicylic acid (SA) and agar solidification of the medium promoted flower maturation and resulted in high flowering rates in L. gibba 7741 and 5504. Artificial cross-pollination between individuals of L. gibba 7741 yielded seeds at high frequencies unlike that in L. gibba 5504. In contrast to clone 7741, the anthers of 5504 did not dehisce upon maturation, its artificially released pollen grains had pineapple-like exine with tilted spines. These pollens were not stained by 2,5-diphenylmonotetrazoliumbromide (MTT) and failed to germinate. Therefore, clone 5504 is male sterile and has potential application with respect to hybrid vigour. Moreover, pollination of flowers of 5504 with 7741 pollen grains resulted in intraspecific hybrid seeds, which was confirmed by inter-simple sequence repeat (ISSR) markers. These hybrid seeds germinated at a high frequency, forming new clones.

West Europe Report, Science and Technology.

DTIC Science & Technology

1986-03-14

Biogen, Under New Leadership, Outlines Recovery Strategy (INDUSTRIEMAGAZIN, 15 Nov 85) 7 Briefs French Artificial Seed Research 13 Transgene ...exceptional wheat plant, one individual can be cloned in thousands of copies within a period of one to three months," re- ports Yves Demarly, director...JPRS-WST-86-009 14 March 1986 ^_ TRANSGENE INCREASES CAPITAL—The French genetic engineering laboratory Transgene , created in 1980 at Parisbas

Citrus psorosis virus coat protein-derived hairpin construct confers stable transgenic resistance in citrus against psorosis A and B syndromes.

PubMed

De Francesco, A; Costa, N; García, M L

2017-04-01

Citrus psorosis virus (CPsV) is the causal agent of psorosis, a serious and widespread citrus disease. Two syndromes of psorosis, PsA and PsB, have been described. PsB is the most aggressive and rampant form. Previously, we obtained Pineapple sweet orange plants transformed with a hairpin construct derived from the CPsV coat protein gene (ihpCP). Some of these plants were resistant to CPsV 90-1-1, a PsA isolate homologous to the transgene. In this study, we found that expression of the ihpCP transgene and siRNA production in lines ihpCP-10 and -15 were stable with time and propagation. In particular, line ihpCP-15 has been resistant for more than 2 years, even after re-inoculation. The ihpCP plants were also resistant against a heterologous CPsV isolate that causes severe PsB syndrome. Line ihpCP-15 manifested complete resistance while line ihpCP-10 was tolerant to the virus, although with variable behaviour, showing delay and attenuation in PsB symptoms. These lines are promising for a biotech product aimed at eradicating psorosis.

[Telomere lengthening by trichostatin A treatment in cloned pigs].

PubMed

Xie, Bing-Teng; Ji, Guang-Zhen; Kong, Qing-Ran; Mao, Jian; Shi, Yong-Qian; Liu, Shi-Chao; Wu, Mei-Ling; Wang, Juan; Liu, Lin; Liu, Zhong-Hua

2012-12-01

Telomeres are repeated GC rich sequences at the end of chromosomes, and shorten with each cell division due to DNA end replication problem. Previously, reprogrammed somatic cells of cloned animals display variable telomere elongation. However, it was reported that the cloned animals including Dolly do not reset telomeres and show premature aging. In this study, we investigated telomere function in cloned or transgenic cloned pigs, including the cloned Northeast Min pigs, eGFP, Mx, and PGC1α transgenic cloned pigs, and found that the telomere lengths of cloned pigs were significantly shorter than the nuclear donor adult fibroblasts and age-matched noncloned pigs (P<0.05), indicating that nuclear reprogramming did not restore cellular age of donor cells after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA), an inhibitor of histone deacetylase, has proven to enhance the efficiency of nuclear reprogramming in several species. In order to test whether TSA also can effectively enhance reprogramming of telomeres, TSA (40 nmol/L) was used to treat porcine cloned embryos at 1-cell stage for 24 h. Consistent with previous reports, the developmental rate of SCNT embryos to the blastocyst stage was significantly increased compared with those of the control group (16.35% vs. 27.09%, 21.60% vs. 34.90%, P<0.05). Notably, the telomere length of cloned porcine blastocysts was also significantly elongated (P<0.05). Although TSA did not improve the cloning efficiency (1.3% vs. 1.7%, TSA vs. control), the telomere lengths of cloned pig-lets were significantly longer compared with those of the control group and the donor fibroblasts (P<0.05). In conclusion, telomeres have not been effectively restored by SCNT in pigs but TSA can effectively lengthen the telomere lengths of cloned pigs.

Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

PubMed

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-03-16

There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

PubMed Central

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-01-01

Background There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale. PMID:21436886

Cloning-free CRISPR

PubMed Central

Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I.

2015-01-01

Summary We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis. PMID:26527385

Big Animal Cloning Using Transgenic Induced Pluripotent Stem Cells: A Case Study of Goat Transgenic Induced Pluripotent Stem Cells.

PubMed

Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Wang, Ziyu; Wang, Feng

2016-02-01

Using of embryonic stem cells (ESCs) could improve production traits and disease resistance by improving the efficiency of somatic cell nuclear transfer (SCNT) technology. However, robust ESCs have not been established from domestic ungulates. In the present study, we generated goat induced pluripotent stem cells (giPSCs) and transgenic cloned dairy goat induced pluripotent stem cells (tgiPSCs) from dairy goat fibroblasts (gFs) and transgenic cloned dairy goat fibroblasts (tgFs), respectively, using lentiviruses that contained hOCT4, hSOX2, hMYC, and hKLF4 without chemical compounds. The giPSCs and tgiPSCs expressed endogenous pluripotent markers, including OCT4, SOX2, MYC, KLF4, and NANOG. Moreover, they were able to maintain a normal karyotype and differentiate into derivatives from all three germ layers in vitro and in vivo. Using SCNT, tgFs and tgiPSCs were used as donor cells to produce embryos, which were named tgF-Embryos and tgiPSC-Embryos. The fusion rates and cleavage rates had no significant differences between tgF-Embryos and tgiPSC-Embryos. However, the expression of IGF-2, which is an important gene associated with embryonic development, was significantly lower in tgiPSC-Embryos than in tgF-Embryos and was not significantly different from vivo-Embryos.

Genomics and Ethics: The Case of Cloned and/or Transgenic Animals

PubMed Central

2003-01-01

The point of the present study is to illustrate and, if possible, promote the existing link between genomics and ethics, taking the example of cloned and transgenic animals. These ‘new animals’ raise theoretical and practical problems that concern applied ethics. We will explore more particularly an original strategy showing that it is possible, starting from philosophical questioning about the nature of identity, to use a genomic approach, based on amplification fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) detection, to provide useful tools to define more rigorously what cloned animals are, by testing their genetic and epigenetic identity. We expect from the future results of this combined approach to stimulate the creativity of the philosophical and ethical reflection about the impact of biotechnology on animals, and to increase scientific involvement in such issues. PMID:18629111

Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple).

PubMed

Antony, Edna; Taybi, Tahar; Courbot, Mikaël; Mugford, Sam T; Smith, J Andrew C; Borland, Anne M

2008-01-01

In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

Production of diabetic offspring using cryopreserved epididymal sperm by in vitro fertilization and intrafallopian insemination techniques in transgenic pigs.

PubMed

Umeyama, Kazuhiro; Honda, Kasumi; Matsunari, Hitomi; Nakano, Kazuaki; Hidaka, Tatsuro; Sekiguchi, Keito; Mochizuki, Hironori; Takeuchi, Yasuhiro; Fujiwara, Tsukasa; Watanabe, Masahito; Nagaya, Masaki; Nagashima, Hiroshi

2013-12-17

Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs.

Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by In Vitro Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

PubMed Central

UMEYAMA, Kazuhiro; HONDA, Kasumi; MATSUNARI, Hitomi; NAKANO, Kazuaki; HIDAKA, Tatsuro; SEKIGUCHI, Keito; MOCHIZUKI, Hironori; TAKEUCHI, Yasuhiro; FUJIWARA, Tsukasa; WATANABE, Masahito; NAGAYA, Masaki; NAGASHIMA, Hiroshi

2013-01-01

Abstract Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs. PMID:23979397

Microarray analysis of gene expression profiles in ripening pineapple fruits.

PubMed

Koia, Jonni H; Moyle, Richard L; Botella, Jose R

2012-12-18

Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general.

Microarray analysis of gene expression profiles in ripening pineapple fruits

PubMed Central

2012-01-01

Background Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Results Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. Conclusions This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general. PMID:23245313

Increasing efficiency in production of cloned piglets.

PubMed

Callesen, Henrik; Liu, Ying; Pedersen, Hanne S; Li, Rong; Schmidt, Mette

2014-12-01

The low efficiency in obtaining piglets after production of cloned embryos was challenged in two steps-first by performing in vitro culture for 5-6 days after cloning to obtain later-stage embryos for more precise selection for transfer, and second by reducing the number of embryos transferred per recipient sow. The data set consisted of combined results from a 4-year period where cloning was performed to produce piglets that were transgenic for important human diseases. For this, different transgenes and cell types were used, and the cloning work was performed by several persons using oocytes from different pig breeds, but following a standardized and optimized protocol. Results showed that in vitro culture is possible with a relatively stable rate of transferable embryos around 41% and a pregnancy rate around 90%. Furthermore, a reduction from around 80 embryos to 40 embryos transferred per recipient was possible without changing the efficiency of around 14% (piglets born out of embryos transferred). It was concluded that this approach can increase the efficiency in obtaining piglets by means of in vitro culture and selection of high-quality embryos with subsequent transfer into more recipients. Such changes can also reduce the need for personnel, time, and material when working with this technology.

Monitoring transgenic plants using in vivo markers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, C.N. Jr.

The gene coding for green fluorecent protein (GFP), isolated and cloned from the jellyfish Aequorea victoria, is an ideal transgene for the monitoring of any plant species. It has the ability to fluoresce without added substrate, enzyme, or cofactor; it does not introduce morphological or sexual aberrations when expressed. 7 refs., 1 fig.

Identification of Short Hairpin RNA Targeting Foot-And-Mouth Disease Virus with Transgenic Bovine Fetal Epithelium Cells

PubMed Central

He, Hongbin; Ding, Fangrong; Yang, Hongjun; Cheng, Lei; Liu, Wenhao; Zhong, Jifeng; Dai, Yunping; Li, Guangpeng; He, Chengqiang; Yu, Li; Li, Jianbin

2012-01-01

Background Although it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. Principal Finding Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. Conclusion RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV. PMID:22905125

Isogenic transgenic homozygous fish induced by artificial parthenogenesis.

PubMed

Nam, Y K; Cho, Y S; Kim, D S

2000-12-01

As a model system for vertebrate transgenesis, fish have many attractive advantages, especially with respect to the characteristics of eggs, allowing us to produce isogenic, transgenic, homozygous vertebrates by combining with chromosome-set manipulation. Here, we describe the large-scale production of isogenic transgenic homozygous animals using our experimental organism, the mud loach Misgurnus mizolepis, by the simple process of artificial parthenogenesis in a single generation. These isogenic fish have retained transgenic homozygous status in a stable manner during the subsequent 5 years, and exhibited increased levels of transgene expression. Furthermore, their isogenic nature was confirmed by cloned transgenic homozygous offspring produced via another step of parthenogenic reproduction of the isogenic homozygous transgenic fish. These results demonstrate that a combination of transgenesis and artificial parthenogenesis will make the rapid utilization of genetically pure homozygous transgenic system in vertebrate transgenesis possible.

Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

PubMed

Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

2013-10-01

This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

[Construction of transgenic tobacco expressing popW and analysis of its biological phenotype].

PubMed

Wang, Cui; Liu, Hongxia; Cao, Jing; Wang, Chao; Guo, Jianhua

2014-04-01

In a previous study, we cloned popW from Ralstonia solanacearum strain ZJ3721, coding PopW, a new harpin protein. The procaryotically expressed PopW can induce resistance to Tobacco mosaic virus (TMV), enhance growth and improve quality of tobacco, when sprayed onto tobacco leaves. Here, we constructed an expression vector pB- popW by cloning popW into the bionary vector pBI121 and transformed it into Agrobacterium tumefaciens strain EHA105 via freeze-thaw method. Tobacco (Nicotiana tobacum cv. Xanthi nc.) transformation was conducted by infection of tobacco leaf discs with recombinant A. tumefaciens. After screening on MS medium containing kanamycin, PCR and RT-PCR analysis, 21 T3 lines were identified as positive transgenic. Genomic intergration and expression of the transferred gene were determined by PCR and RT-PCR. And GUS staining analysis indicated that the protein expressed in transgenic tobacco was bioactive and exhibited different expression levels among lines. Disease bioassays showed that the transgenic tobacco had enhanced resistance to TMV with biocontrol efficiency up to 54.25%. Transgenic tobacco also exhibited enhanced plant growth, the root length of 15 d old seedlings was 1.7 times longer than that of wild type tobacco. 60 d after transplanting to pots, the height, fresh weight and dry weight of transgenic tobacco were 1.4, 1.7, 1.8 times larger than that of wild type tobacco, respectively.

Isolation and characterization of a FLOWERING LOCUS T homolog from pineapple (Ananas comosus (L.) Merr).

PubMed

Lv, LingLing; Duan, Jun; Xie, JiangHui; Wei, ChangBin; Liu, YuGe; Liu, ShengHui; Sun, GuangMing

2012-09-01

FLOWERING LOCUS T (FT)-like genes are crucial regulators of flowering in angiosperms. A homolog of FT, designated as AcFT (GenBank ID: HQ343233), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcFT is 915 bp in length and contains an ORF of 534 bp, which encodes a protein of 177 aa. Molecular weight was 19.9 kDa and isoelectric point was 6.96. The deduced protein sequence of AcFT was 84% and 82% identical to homologs encoded by CgFT in Cymbidium goeringii and OgFT in Oncidium Gower Ramsey respectively. Quantitative real-time PCR (qRT-PCR) analyses showed that the expression of AcFT was high in flesh and none in leaves. qRT-PCR analyses in different stages indicated that the expression of AcFT reached the highest level on 40 d after flower inducing, when the multiple fruit and floral organs were forming. The 35S::AcFT transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants. Copyright © 2012 Elsevier B.V. All rights reserved.

Functional characterization of enone oxidoreductases from strawberry and tomato fruit.

PubMed

Klein, Dorothée; Fink, Barbara; Arold, Beate; Eisenreich, Wolfgang; Schwab, Wilfried

2007-08-08

Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxidoreductase, is a ripening-induced, negatively auxin-regulated enzyme that catalyzes the formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), the key flavor compound in strawberry fruit by the reduction of the alpha,beta-unsaturated bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF). Here we show that recombinant FaEO does not reduce the double bond of straight-chain 2-alkenals or 2-alkenones but rather hydrogenates previously unknown HMMF derivatives substituted at the methylene functional group. The furanones were prepared from 4-hydroxy-5-methyl-3(2H)-furanone with a number of aldehydes and a ketone. The kinetic data for the newly synthesized aroma-active substrates and products are similar to the values obtained for an enone oxidoreductase from Arabidopsis thaliana catalyzing the alpha,beta-hydrogenation of 2-alkenals. HMMF, the substrate of FaEO that is formed during strawberry fruit ripening, was also detected in tomato and pineapple fruit by HPLC-ESI-MSn and became 13C-labeled when d-[6-13C]-glucose was applied to the fruits, which suggested that a similar HDMF biosynthetic pathway occurs in the different plant species. With a database search (http://ted.bti.cornell.edu/ and http://genet.imb.uq.edu.au/Pineapple/), we identified a tomato and pineapple expressed sequence tag that shows significant homology to FaEO. Solanum lycopersicon EO (SlEO) was cloned from cDNA, and the protein was expressed in Escherichia coli and purified. Biochemical studies confirmed the involvement of SlEO in the biosynthesis of HDMF in tomato fruit.

Drought-tolerant rice germplasm developed from an Oryza officinalis transformation-competent artificial chromosome clone.

PubMed

Liu, R; Zhang, H H; Chen, Z X; Shahid, M Q; Fu, X L; Liu, X D

2015-10-29

Oryza officinalis has proven to be a natural gene reservoir for the improvement of domesticated rice as it carries many desirable traits; however, the transfer of elite genes to cultivated rice by conventional hybridization has been a challenge for rice breeders. In this study, the conserved sequence of plant stress-related NAC transcription factors was selected as a probe to screen the O. officinalis genomic transformation-competent artificial chromosome library by Southern blot; 11 positive transformation-competent artificial chromosome clones were subsequently detected. By Agrobacterium-mediated transformation, an indica rice variety, Huajingxian 74 (HJX74), was transformed with a TAC clone harboring a NAC gene-positive genomic fragment from O. officinalis. Molecular analysis revealed that the O. officinalis genomic fragment was integrated into the genome of HJX74. The transgenic lines exhibited high tolerance to drought stress. Our results demonstrate that the introduction of stress-related transformation-competent artificial chromosome clones, coupled with a transgenic validation approach, is an effective method of transferring agronomically important genes from O. officinalis to cultivated rice.

The dominant expression of functional human lactoferrin in transgenic cloned goats using a hybrid lactoferrin expression construct.

PubMed

Yu, Huiqing; Chen, Jianquan; Sun, Wei; Liu, Siguo; Zhang, Aimin; Xu, Xujun; Wang, Xuebin; He, Zhuzi; Liu, Guohui; Cheng, Guoxiang

2012-10-31

Human Lactoferrin (hLF) is an iron-binding protein with multiple physiological functions. As the availability of natural hLF is limited, alternative means of producing this biopharmaceutical protein have been extensively studied. Here we report on the dominant expression of recombinant human lactoferrin (rhLF) in transgenic cloned goats using a novel optimised construct made by fusing a 3.3 kb hLF minigene to the regulatory elements of the β-casein gene. The transgenic goat produced more than 30 mg/ml rhLF in its milk, and rhLF expression was stable during the entire lactation cycle. The rhLF purification efficiency from whole goat milk is approximately 70%, and its purity is above 98%. Compared with natural hLF, the rhLF from transgenic goats has similar biological characteristics including molecular mass, N-terminal sequence, isoelectric point, immunoreactivity and digestive stability. More importantly, the purified rhLF showed specific anti-tumour activity in the mouse model of melanoma experimental metastasis. Therefore, our study shows that the large-scale production of functional rhLF in transgenic goat milk could be an economical and promising source of human therapeutic use in the future. Copyright © 2012. Published by Elsevier B.V.

Insertional Mutagenesis for Genes involved in Otic/Vestibular Development and Function in Xenopus Tropicalis

NASA Technical Reports Server (NTRS)

Torrejon, Marcela; Li, Erica; Nguyen, Minh; Winfree, Seth; Wang, Esther; Reinsch, Sigrid; Dalton, Bonnie (Technical Monitor)

2002-01-01

Sensitivity to gravity is essential for spatial orientation. Consequently, the gravity receptor system is one of the phylogenetically oldest sensory systems, and the special adaptations that enhance sensitivity to gravity are highly conserved. The main goal of this project is to use Xenopus (frog) to identify genes expressed during vestibular and auditory development. These studies will lead a better understanding of the molecular mechanisms involved in vestibular and auditory development and function. We are using a gene-trap approach in Xenopus tropicalis with the green fluorescent protein (GFP) gene as the transgene reporter. GFP expression occurs only when the GFP gene is correctly integrated in actively transcribed genes. Using the GFP as a tag we can easily identify and clone the mutated gene. In addition, we can study the function of the mutated gene by analyzing the defects generated by insertion of the GFP transgene. To date we have tissue specific GFP expression in X. tropicalis including expression in ear, neural tube, kidney, muscle, eyes and nose. Our transgenic animals will soon reach maturity so that we can outcross them and analyze their progeny. Our next goal is to isolate RNA from our transgenics and clone the tagged genes using RACE-PCR. Currently we are optimizing the RACE-PCR method using transgenics with crystallin GFP expression.

Technologies for Hemostasis and Stabilization of the Acute Traumatic Wound

DTIC Science & Technology

2012-10-01

Fibrinogen Produced in the Milk of Transgenic Cows ” (see abstract in Appendix A10). 4. A poster presentation “Development of Novel Hemostatic Devices in...biosynthesis and material characterization of rFI made in the milk of cloned, transgenic dairy cows . To our knowledge, rFI is one of the most challenging...rFI was purified from 32 liters of milk collected on eight lactation days from two transgenic cows (Foxy and Fantasy) of the BFI2n8c83-EGFIneo

US SEEDLESS PINEAPPLE ORANGE

USDA-ARS?s Scientific Manuscript database

The Agricultural Research Service, U.S. Department of Agriculture, hereby releases for propagation the US SEEDLESS PINEAPPLE citrus scion selection, formerly tested as USDA 1-10-60. US SEEDLESS PINEAPPLE resulted from irradiation of Ridge Pineapple seeds by C.J. Hearn in 1970 at the U.S. Horticultu...

Phylogeography of pink pineapple mealybugs, Dysmicoccus brevipes (Cockerell) reveals the history of pineapple introduction and cultivation in China.

PubMed

He, Y B; Zhan, R L; Sun, G M; Wu, J B; Zhao, Y L

2015-08-19

The pink pineapple mealybug (PPM), Dysmicoccus brevipes (Cockerell) (Hemiptera: Pseudococcidae), is a widespread plant-sucking insect of considerable concern because it transmits the pineapple mealybug wilt-associated virus. Its distribution is closely linked with its host, the pineapple [Ananas comosus (L.) Merrill] because of its wingless and parthenogenetic characteristics. To investigate the history of D. brevipes introduction and the cultivation of pineapple in China, samples of D. brevipes were collected from the main pineapple production region in China, and from Thailand, and the mitochondrial cytochrome c oxidase subunit I (COI) gene was analyzed. Homologous sequences of D. brevipes COI from Brazil, Thailand, and Philippines that are deposited in GenBank were compared. Phylogenetic analyses suggest there are close genetic relationships between PPM populations from Hawaii, Brazil, the Philippines, and from Thailand and China, which probably originate from South America. It is suggested that most PPMs in China were introduced from South America by way of Southeast Asia, being accompanied by the pineapple seedling. Conversely, some PPMs represented by Haplotype-WN from Wanning of China, and Lampang of Thailand were found to differ greatly from populations in Hawaii, Brazil, the Philippines, Thailand, and China. It is possible that another route was used for the introduction and distribution of pineapple, or that pineapple might have originated in Southeast Asia.

Developing single nucleotide polymorphism markers for the identification of pineapple (Ananas comosus) germplasm.

PubMed

Zhou, Lin; Matsumoto, Tracie; Tan, Hua-Wei; Meinhardt, Lyndel W; Mischke, Sue; Wang, Boyi; Zhang, Dapeng

2015-01-01

Pineapple (Ananas comosus [L.] Merr.) is the third most important tropical fruit in the world after banana and mango. As a crop with vegetative propagation, genetic redundancy is a major challenge for efficient genebank management and in breeding. Using expressed sequence tag and nucleotide sequences from public databases, we developed 213 single nucleotide polymorphism (SNP) markers and validated 96 SNPs by genotyping the United States Department of Agriculture - Agricultural Research Service pineapple germplasm collection, maintained in Hilo, Hawaii. The validation resulted in designation of a set of 57 polymorphic SNP markers that revealed a high rate of duplicates in this pineapple collection. Twenty-four groups of duplicates were detected, encompassing 130 of the total 170 A cosmos accessions. The results show that somatic mutation has been the main source of intra-cultivar variations in pineapple. Multivariate clustering and a model-based population stratification suggest that the modern pineapple cultivars are comprised of progenies that are derived from different wild Ananas botanical varieties. Parentage analysis further revealed that both A. comosus var. bracteatus and A. comosus var. ananassoides are likely progenitors of pineapple cultivars. However, the traditional classification of cultivated pineapple into horticultural groups (e.g. 'Cayenne', 'Spanish', 'Queen') was not well supported by the present study. These SNP markers provide robust and universally comparable DNA fingerprints; thus, they can serve as an efficient genotyping tool to assist pineapple germplasm management, propagation of planting material, and pineapple cultivar protection. The high rate of genetic redundancy detected in this pineapple collection suggests the potential impact of applying this technology on other clonally propagated perennial crops.

A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin.

PubMed

Watanabe, Satoshi; Iwamoto, Masaki; Suzuki, Shun-ichi; Fuchimoto, Daiichiro; Honma, Daisuke; Nagai, Takashi; Hashimoto, Michiko; Yazaki, Satoko; Sato, Masahiro; Onishi, Akira

2005-02-01

Puromycin N-acetyl transferase gene (pac), of which the gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in embryonic stem (ES) cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of enhanced green fluorescent protein (EGFP) transgenic piglets after somatic cell cloning and embryo transfer. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene (pCAG-EGFPac) carrying both EGFP cDNA and pac. This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 microg/ml), cultured for 7 days, and then screened for EGFP expression before somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and nine piglets were delivered. Of the nine piglets, eight died shortly after birth and one grew healthy after weaning. Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.

Bioactive compounds and antioxidant activity analysis of Malaysian pineapple cultivars

NASA Astrophysics Data System (ADS)

Chiet, Chong Hang; Zulkifli, Razauden Mohamed; Hidayat, Topik; Yaakob, Harisun

2014-03-01

Pineapple industry is one of the important agricultural sectors in Malaysia with 76 cultivars planted throughout the country. This study aims to generate useful nutritional information as well as evaluating antioxidant properties of different pineapple commercial cultivars in Malaysia. The bioactive compound content and antioxidant capacity of `Josapine', `Morris' and `Sarawak' pineapple (Ananas comosus) were studied. The pineapple varieties were collected at commercial maturity stage (20-40% yellowish of fruit peel) and the edible portion of the fruit was used as sample for evaluation. The bioactive compound of the fruit extracts were evaluated by total phenolic and tannin content assay while the antioxidant capacity was determined by ferric reducing antioxidant power (FRAP). From the results obtained, total phenolic and tannin content was highest for `Josapine' followed by `Morris' and `Sarawak'. With respect to FRAP, `Josapine' showed highest reducing capacity, followed by `Morris' and then `Sarawak' having the least value. The bioactive compounds content are positively correlated with the antioxidant capacities of the pineapple extracts. This result indicates that the total phenolics and tannin content present in the pineapples may contribute to the antioxidant capacity of the pineapples.

Enhanced pest resistance and increased phenolic production in maize callus transgenically expressing a maize chalcone isomerase -3 like gene

USDA-ARS?s Scientific Manuscript database

Significant losses in maize production are due to damage by insects and ear rot fungi. A gene designated as chalcone-isomerase-like, located in a quantitative trait locus for resistance to Fusarium ear rot fungi, was cloned from a Fusarium ear rot resistant inbred and transgenically expressed in mai...

Developing single nucleotide polymorphism markers for the identification of pineapple (Ananas comosus) germplasm

PubMed Central

Zhou, Lin; Matsumoto, Tracie; Tan, Hua-Wei; Meinhardt, Lyndel W; Mischke, Sue; Wang, Boyi; Zhang, Dapeng

2015-01-01

Pineapple (Ananas comosus [L.] Merr.) is the third most important tropical fruit in the world after banana and mango. As a crop with vegetative propagation, genetic redundancy is a major challenge for efficient genebank management and in breeding. Using expressed sequence tag and nucleotide sequences from public databases, we developed 213 single nucleotide polymorphism (SNP) markers and validated 96 SNPs by genotyping the United States Department of Agriculture - Agricultural Research Service pineapple germplasm collection, maintained in Hilo, Hawaii. The validation resulted in designation of a set of 57 polymorphic SNP markers that revealed a high rate of duplicates in this pineapple collection. Twenty-four groups of duplicates were detected, encompassing 130 of the total 170 A cosmos accessions. The results show that somatic mutation has been the main source of intra-cultivar variations in pineapple. Multivariate clustering and a model-based population stratification suggest that the modern pineapple cultivars are comprised of progenies that are derived from different wild Ananas botanical varieties. Parentage analysis further revealed that both A. comosus var. bracteatus and A. comosus var. ananassoides are likely progenitors of pineapple cultivars. However, the traditional classification of cultivated pineapple into horticultural groups (e.g. ‘Cayenne’, ‘Spanish’, ‘Queen’) was not well supported by the present study. These SNP markers provide robust and universally comparable DNA fingerprints; thus, they can serve as an efficient genotyping tool to assist pineapple germplasm management, propagation of planting material, and pineapple cultivar protection. The high rate of genetic redundancy detected in this pineapple collection suggests the potential impact of applying this technology on other clonally propagated perennial crops. PMID:26640697

Cloning of TPS gene from eelgrass species Zostera marina and its functional identification by genetic transformation in rice.

PubMed

Zhao, Feng; Li, Qiuying; Weng, Manli; Wang, Xiuliang; Guo, Baotai; Wang, Li; Wang, Wei; Duan, Delin; Wang, Bin

2013-12-01

The full-length cDNA sequence (2613 bp) of the trehalose-6-phosphate synthase (TPS) gene of eelgrass Zostera marina (ZmTPS) was identified and cloned. Z. marina is a kind of seed-plant growing in sea water during its whole life history. The open reading frame (ORF) region of ZmTPS gene encodes a protein of 870 amino acid residues and a stop codon. The corresponding genomic DNA sequence is 3770 bp in length, which contains 3 exons and 2 introns. The ZmTPS gene was transformed into rice variety ZH11 via Agrobacterium-mediated transformation method. After antibiotic screening, molecular characterization, salt-tolerance and trehalose content determinations, two transgenic lines resistant to 150 mM NaCL solutions were screened. Our study results indicated that the ZmTPS gene was integrated into the genomic DNA of the two transgenic rice lines and could be expressed well. Moreover, the detection of the transformed ZmTPS gene in the progenies of the two transgenic lines was performed from T1 to T4 generations; and results suggested that the transformed ZmTPS gene can be transmitted from parent to the progeny in transgenic rice. © 2013.

Wheat TaSP gene improves salt tolerance in transgenic Arabidopsis thaliana.

PubMed

Ma, Xiaoli; Cui, Weina; Liang, Wenji; Huang, Zhanjing

2015-12-01

A novel salt-induced gene with unknown functions was cloned through analysis of gene expression profile of a salt-tolerant wheat mutant RH8706-49 under salt stress. The gene was named Triticum aestivum salt-related protein (TaSP) and deposited in GenBank (Accession No. KF307326). Quantitative polymerase chain reaction (qPCR) results showed that TaSP expression was induced under salt, abscisic acid (ABA), and polyethylene glycol (PEG) stresses. Subcellular localization revealed that TaSP was mainly localized in cell membrane. Overexpression of TaSP in Arabidopsis could improve salt tolerance of 35S::TaSP transgenic Arabidopsis. 35S::TaSP transgenic Arabidopsis lines after salt stress presented better physiological indexes than the control group. In the non-invasive micro-test (NMT), an evident Na(+) excretion was observed at the root tip of salt-stressed 35S::TaSP transgenic Arabidopsis. TaSP promoter was cloned, and its beta-glucuronidase (GUS) activities before and after ABA, salt, cold, heat, and salicylic acid (SA) stresses were determined. Full-length TaSP promoter contained ABA and salt response elements. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Production of cloned transgenic cow expressing omega-3 fatty acids.

PubMed

Wu, Xia; Ouyang, Hongsheng; Duan, Biao; Pang, Daxin; Zhang, Li; Yuan, Ting; Xue, Lian; Ni, Daibang; Cheng, Lei; Dong, Shuhua; Wei, Zhuying; Li, Lin; Yu, Ming; Sun, Qing-Yuan; Chen, Da-Yuan; Lai, Liangxue; Dai, Yifan; Li, Guang-Peng

2012-06-01

n-3 Polyunsaturated fatty acids (n-3 PUFA) are important for human health. Alternative resources of n-3 PUAFs created by transgenic domestic animals would be an economic approach. In this study, we generated a mfat-1 transgenic cattle expressed a Caenorhabditis elegans gene, mfat-1, encoding an n-3 fatty acid desaturase. Fatty acids analysis of tissue and milk showed that all of the examined n-3 PUAFs were greatly increased and simultaneously the n-6 PUAFs decreased in the transgenic cow. A significantly reduction of n-6/n-3 ratios (P<0.05) in both tissue and milk were observed.

Production of transgenic-cloned pigs expressing large quantities of recombinant human lysozyme in milk.

PubMed

Lu, Dan; Liu, Shen; Shang, Shengzhe; Wu, Fangfang; Wen, Xiao; Li, Zhiyuan; Li, Yan; Hu, Xiaoxiang; Zhao, Yaofeng; Li, Qiuyan; Li, Ning

2015-01-01

Human lysozyme is a natural non-specific immune factor in human milk that plays an important role in the defense of breastfed infants against pathogen infection. Although lysozyme is abundant in human milk, there is only trace quantities in pig milk. Here, we successfully generated transgenic cloned pigs with the expression vector pBAC-hLF-hLZ-Neo and their first generation hybrids (F1). The highest concentration of recombinant human lysozyme (rhLZ) with in vitro bioactivity was 2759.6 ± 265.0 mg/L in the milk of F0 sows. Compared with wild-type milk, rhLZ milk inhibited growth of Escherichia coli K88 during the exponential growth phase. Moreover, rhLZ in milk from transgenic sows was directly absorbed by the intestine of piglets with no observable anaphylactic reaction. Our strategy may provide a powerful tool for large-scale production of this important human protein in pigs to improve resistance to pathogen infection.

Immunogenicity of recombinant LT-B delivered orally to humans in transgenic corn.

PubMed

Tacket, Carol O; Pasetti, Marcela F; Edelman, Robert; Howard, John A; Streatfield, Stephen

2004-10-22

Previous clinical studies have demonstrated the feasibility of using edible transgenic plants to deliver protective antigens as new oral vaccines. Transgenic corn is particularly attractive for this purpose since the recombinant antigen is stable and homogeneous, and corn can be formulated in several edible forms without destroying the cloned antigen. Transgenic corn expressing 1 mg of LT-B of Escherichia coli without buffer was fed to adult volunteers in three doses, each consisting of 2.1 g of plant material. Seven (78%) of nine volunteers developed rises in both serum IgG anti-LT and numbers of specific antibody secreting cells after vaccination. Four (44%) of nine volunteers also developed stool IgA. Transgenic plants represent a new vector for oral vaccine antigens.

Direct introduction of gene constructs into the pronucleus-like structure of cloned embryos: a new strategy for the generation of genetically modified pigs.

PubMed

Kurome, Mayuko; Leuchs, Simon; Kessler, Barbara; Kemter, Elisabeth; Jemiller, Eva-Maria; Foerster, Beatrix; Klymiuk, Nikolai; Zakhartchenko, Valeri; Wolf, Eckhard

2017-04-01

Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed "nuclear injection". To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.

Molecular cloning and functional analysis of the drought tolerance gene MsHSP70 from alfalfa (Medicago sativa L.).

PubMed

Li, Zhenyi; Long, Ruicai; Zhang, Tiejun; Wang, Zhen; Zhang, Fan; Yang, Qingchuan; Kang, Junmei; Sun, Yan

2017-03-01

Heat shock proteins (HSPs) are a ubiquitously expressed class of protective proteins that play a key role in plant response to stressful conditions. This study aimed to characterize and investigate the function of an HSP gene in alfalfa (Medicago sativa). MsHSP70, which contains a 2028-bp open reading frame, was identified through homology cloning. MsHSP70 shares high sequence identity (94.47%) with HSP70 from Medicago truncatula. Expression analysis of MsHSP70 in alfalfa organs revealed a relatively higher expression level in aerial organs such as flowers, stems and leaves than in roots. MsHSP70 was induced by heat shock, abscisic acid (ABA) and hydrogen peroxide. Transgenic Arabidopsis seedlings overexpressing MsHSP70 were hyposensitive to polyethylene glycol (PEG) and ABA treatments, suggesting that exogenous expression of MsHSP70 enhanced Arabidopsis tolerance to these stresses. Examination of physiological indexes related to drought and ABA stress demonstrated that in comparison with non-transgenic plants, T3 transgenic Arabidopsis plants had an increased proline content, higher superoxide dismutase (SOD) activity, and decreased malondialdehyde (MDA) content. Furthermore, higher relative water content (RWC) was detected in transgenic plants compared with non-transgenic plants under drought stress. These findings clearly indicate that molecular manipulation of MsHSP70 in plants can have substantial effects on stress tolerance.

Identification and functional characterization of the soybean GmaPPO12 promoter conferring Phytophthora sojae induced expression.

PubMed

Chai, Chunyue; Lin, Yanling; Shen, Danyu; Wu, Yuren; Li, Hongjuan; Dou, Daolong

2013-01-01

Identification of pathogen-inducible promoters largely lags behind cloning of the genes for disease resistance. Here, we cloned the soybean GmaPPO12 gene and found that it was rapidly and strongly induced by Phytophthorasojae infection. Computational analysis revealed that its promoter contained many known cis-elements, including several defense related transcriptional factor-binding boxes. We showed that the promoter could mediate induction of GUS expression upon infection in both transient expression assays in Nicotianabenthamiana and stable transgenic soybean hairy roots. Importantly, we demonstrated that pathogen-induced expression of the GmaPPO12 promoter was higher than that of the soybean GmaPR1a promoter. A progressive 5' and 3' deletion analysis revealed two fragments that were essential for promoter activity. Thus, the cloned promoter could be used in transgenic plants to enhance resistance to phytophthora pathogens, and the identified fragment could serve as a candidate to produce synthetic pathogen-induced promoters.

Field Performance of Transgenic Sugarcane Lines Resistant to Sugarcane Mosaic Virus

PubMed Central

Yao, Wei; Ruan, Miaohong; Qin, Lifang; Yang, Chuanyu; Chen, Rukai; Chen, Baoshan; Zhang, Muqing

2017-01-01

Sugarcane mosaic disease is mainly caused by the sugarcane mosaic virus (SCMV), which can significantly reduce stalk yield and sucrose content of sugarcane in the field. Coat protein mediated protection (CPMP) is an effective strategy to improve virus resistance. A 2-year field study was conducted to compare five independent transgenic sugarcane lines carrying the SCMV-CP gene (i.e., B2, B36, B38, B48, and B51) with the wild-type parental clone Badila (WT). Agronomic performance, resistance to SCMV infection, and transgene stability were evaluated and compared with the wild-type parental clone Badila (WT) at four experimental locations in China across two successive seasons, i.e., plant cane (PC) and 1st ratoon cane (1R). All transgenic lines derived from Badila had significantly greater tons of cane per hectare (TCH) and tons of sucrose per hectare (TSH) as well as lower SCMV disease incidence than those from Badila in the PC and 1R crops. The transgenic line B48 was highly resistant to SCMV with less than 3% incidence of infection. The recovery phenotype of transgenic line B36 was infected soon after virus inoculation, but the subsequent leaves showed no symptoms of infection. Most control plants developed symptoms that persisted and spread throughout the plant with more than 50% incidence. B48 recorded an average of 102.72 t/ha, which was 67.2% more than that for Badila. The expression of the transgene was stable over many generations with vegetative propagation. These results show that SCMV-resistant transgenic lines derived from Badila can provide resistant germplasm for sugarcane breeding and can also be used to study virus resistance mechanisms. This is the first report on the development and field performance of transgenic sugarcane plants that are resistant to SCMV infection in China. PMID:28228765

Field Performance of Transgenic Sugarcane Lines Resistant to Sugarcane Mosaic Virus.

PubMed

Yao, Wei; Ruan, Miaohong; Qin, Lifang; Yang, Chuanyu; Chen, Rukai; Chen, Baoshan; Zhang, Muqing

2017-01-01

Sugarcane mosaic disease is mainly caused by the sugarcane mosaic virus (SCMV), which can significantly reduce stalk yield and sucrose content of sugarcane in the field. Coat protein mediated protection (CPMP) is an effective strategy to improve virus resistance. A 2-year field study was conducted to compare five independent transgenic sugarcane lines carrying the SCMV-CP gene (i.e., B2, B36, B38, B48, and B51) with the wild-type parental clone Badila (WT). Agronomic performance, resistance to SCMV infection, and transgene stability were evaluated and compared with the wild-type parental clone Badila (WT) at four experimental locations in China across two successive seasons, i.e., plant cane (PC) and 1st ratoon cane (1R). All transgenic lines derived from Badila had significantly greater tons of cane per hectare (TCH) and tons of sucrose per hectare (TSH) as well as lower SCMV disease incidence than those from Badila in the PC and 1R crops. The transgenic line B48 was highly resistant to SCMV with less than 3% incidence of infection. The recovery phenotype of transgenic line B36 was infected soon after virus inoculation, but the subsequent leaves showed no symptoms of infection. Most control plants developed symptoms that persisted and spread throughout the plant with more than 50% incidence. B48 recorded an average of 102.72 t/ha, which was 67.2% more than that for Badila. The expression of the transgene was stable over many generations with vegetative propagation. These results show that SCMV-resistant transgenic lines derived from Badila can provide resistant germplasm for sugarcane breeding and can also be used to study virus resistance mechanisms. This is the first report on the development and field performance of transgenic sugarcane plants that are resistant to SCMV infection in China.

Genome-Wide Identification and Expression Profiling of ATP-Binding Cassette (ABC) Transporter Gene Family in Pineapple (Ananas comosus (L.) Merr.) Reveal the Role of AcABCG38 in Pollen Development

PubMed Central

Chen, Piaojuan; Li, Yi; Zhao, Lihua; Hou, Zhimin; Yan, Maokai; Hu, Bingyan; Liu, Yanhui; Azam, Syed Muhammad; Zhang, Ziyan; Rahman, Zia ur; Liu, Liping; Qin, Yuan

2017-01-01

Pineapple (Ananas comosus L.) cultivation commonly relies on asexual reproduction which is easily impeded by many factors in agriculture production. Sexual reproduction might be a novel approach to improve the pineapple planting. However, genes controlling pineapple sexual reproduction are still remain elusive. In different organisms a conserved superfamily proteins known as ATP binding cassette (ABC) participate in various biological processes. Whereas, till today the ABC gene family has not been identified in pineapple. Here 100 ABC genes were identified in the pineapple genome and grouped into eight subfamilies (5 ABCAs, 20 ABCBs, 16 ABCCs, 2 ABCDs, one ABCEs, 5 ABCFs, 42 ABCGs and 9 ABCIs). Gene expression profiling revealed the dynamic expression pattern of ABC gene family in various tissues and different developmental stages. AcABCA5, AcABCB6, AcABCC4, AcABCC7, AcABCC9, AcABCG26, AcABCG38 and AcABCG42 exhibited preferential expression in ovule and stamen. Over-expression of AcABCG38 in the Arabidopsis double mutant abcg1-2abcg16-2 partially restored its pollen abortion defects, indicating that AcABCG38 plays important roles in pollen development. Our study on ABC gene family in pineapple provides useful information for developing sexual pineapple plantation which could be utilized to improve pineapple agricultural production. PMID:29312399

Genome-Wide Identification and Expression Profiling of ATP-Binding Cassette (ABC) Transporter Gene Family in Pineapple (Ananas comosus (L.) Merr.) Reveal the Role of AcABCG38 in Pollen Development.

PubMed

Chen, Piaojuan; Li, Yi; Zhao, Lihua; Hou, Zhimin; Yan, Maokai; Hu, Bingyan; Liu, Yanhui; Azam, Syed Muhammad; Zhang, Ziyan; Rahman, Zia Ur; Liu, Liping; Qin, Yuan

2017-01-01

Pineapple ( Ananas comosus L .) cultivation commonly relies on asexual reproduction which is easily impeded by many factors in agriculture production. Sexual reproduction might be a novel approach to improve the pineapple planting. However, genes controlling pineapple sexual reproduction are still remain elusive. In different organisms a conserved superfamily proteins known as ATP binding cassette (ABC) participate in various biological processes. Whereas, till today the ABC gene family has not been identified in pineapple. Here 100 ABC genes were identified in the pineapple genome and grouped into eight subfamilies (5 ABCAs , 20 ABCB s, 16 ABCCs , 2 ABCDs , one ABCEs , 5 ABCFs , 42 ABCGs and 9 ABCIs ). Gene expression profiling revealed the dynamic expression pattern of ABC gene family in various tissues and different developmental stages. AcABCA5, AcABCB6, AcABCC4 , AcABCC7 , AcABCC9 , AcABCG26 , AcABCG38 and AcABCG42 exhibited preferential expression in ovule and stamen. Over-expression of AcABCG38 in the Arabidopsis double mutant abcg1-2abcg16-2 partially restored its pollen abortion defects, indicating that AcABCG38 plays important roles in pollen development. Our study on ABC gene family in pineapple provides useful information for developing sexual pineapple plantation which could be utilized to improve pineapple agricultural production.

Effects of field-grown genetically modified Zoysia grass on bacterial community structure.

PubMed

Lee, Yong-Eok; Yang, Sang-Hwan; Bae, Tae-Woong; Kang, Hong-Gyu; Lim, Pyung-Ok; Lee, Hyo-Yeon

2011-04-01

Herbicide-tolerant Zoysia grass has been previously developed through Agrobacterium-mediated transformation. We investigated the effects of genetically modified (GM) Zoysia grass and the associated herbicide application on bacterial community structure by using culture-independent approaches. To assess the possible horizontal gene transfer (HGT) of transgenic DNA to soil microorganisms, total soil DNAs were amplified by PCR with two primer sets for the bar and hpt genes, which were introduced into the GM Zoysia grass by a callus-type transformation. The transgenic genes were not detected from the total genomic DNAs extracted from 1.5 g of each rhizosphere soils of GM and non-GM Zoysia grasses. The structures and diversities of the bacterial communities in rhizosphere soils of GM and non-GM Zoysia grasses were investigated by constructing 16S rDNA clone libraries. Classifier, provided in the RDP II, assigned 100 clones in the 16S rRNA gene sequences library into 11 bacterial phyla. The most abundant phyla in both clone libraries were Acidobacteria and Proteobacteria. The bacterial diversity of the GM clone library was lower than that of the non- GM library. The former contained four phyla, whereas the latter had seven phyla. Phylogenetic trees were constructed to confirm these results. Phylogenetic analyses of the two clone libraries revealed considerable difference from each other. The significance of difference between clone libraries was examined with LIBSHUFF statistics. LIBSHUFF analysis revealed that the two clone libraries differed significantly (P〈0.025), suggesting alterations in the composition of the microbial community associated with GM Zoysia grass.

A highly sensitive single-tube nested PCR assay for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2)

USDA-ARS?s Scientific Manuscript database

An assay was developed for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2), an important factor in the etiology of mealybug wilt of pineapple. The assay combines reverse transcription of RNA isolated from pineapple with a specific and very sensitive, single, closed-tube nested ...

Odor-active constituents in fresh pineapple (Ananas comosus [L.] Merr.) by quantitative and sensory evaluation.

PubMed

Tokitomo, Yukiko; Steinhaus, Martin; Büttner, Andrea; Schieberle, Peter

2005-07-01

By application of aroma extract dilution analysis (AEDA) to an aroma distillate prepared from fresh pineapple using solvent-assisted flavor evaporation (SAFE), 29 odor-active compounds were detected in the flavor dilution (FD) factor range of 2 to 4,096. Quantitative measurements performed by stable isotope dilution assays (SIDA) and a calculation of odor activity values (OAVs) of 12 selected odorants revealed the following compounds as key odorants in fresh pineapple flavor: 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDF; sweet, pineapple-like, caramel-like), ethyl 2-methylpropanoate (fruity), ethyl 2-methylbutanoate (fruity) followed by methyl 2-methylbutanoate (fruity, apple-like) and 1-(E,Z)-3,5-undecatriene (fresh, pineapple-like). A mixture of these 12 odorants in concentrations equal to those in the fresh pineapple resulted in an odor profile similar to that of the fresh juice. Furthermore, the results of omission tests using the model mixture showed that HDF and ethyl 2-methylbutanoate are character impact odorants in fresh pineapple.

The pineapple genome and the evolution of CAM photosynthesis.

PubMed

Ming, Ray; VanBuren, Robert; Wai, Ching Man; Tang, Haibao; Schatz, Michael C; Bowers, John E; Lyons, Eric; Wang, Ming-Li; Chen, Jung; Biggers, Eric; Zhang, Jisen; Huang, Lixian; Zhang, Lingmao; Miao, Wenjing; Zhang, Jian; Ye, Zhangyao; Miao, Chenyong; Lin, Zhicong; Wang, Hao; Zhou, Hongye; Yim, Won C; Priest, Henry D; Zheng, Chunfang; Woodhouse, Margaret; Edger, Patrick P; Guyot, Romain; Guo, Hao-Bo; Guo, Hong; Zheng, Guangyong; Singh, Ratnesh; Sharma, Anupma; Min, Xiangjia; Zheng, Yun; Lee, Hayan; Gurtowski, James; Sedlazeck, Fritz J; Harkess, Alex; McKain, Michael R; Liao, Zhenyang; Fang, Jingping; Liu, Juan; Zhang, Xiaodan; Zhang, Qing; Hu, Weichang; Qin, Yuan; Wang, Kai; Chen, Li-Yu; Shirley, Neil; Lin, Yann-Rong; Liu, Li-Yu; Hernandez, Alvaro G; Wright, Chris L; Bulone, Vincent; Tuskan, Gerald A; Heath, Katy; Zee, Francis; Moore, Paul H; Sunkar, Ramanjulu; Leebens-Mack, James H; Mockler, Todd; Bennetzen, Jeffrey L; Freeling, Michael; Sankoff, David; Paterson, Andrew H; Zhu, Xinguang; Yang, Xiaohan; Smith, J Andrew C; Cushman, John C; Paull, Robert E; Yu, Qingyi

2015-12-01

Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water-use efficiency, and the second most important tropical fruit. We sequenced the genomes of pineapple varieties F153 and MD2 and a wild pineapple relative, Ananas bracteatus accession CB5. The pineapple genome has one fewer ancient whole-genome duplication event than sequenced grass genomes and a conserved karyotype with seven chromosomes from before the ρ duplication event. The pineapple lineage has transitioned from C3 photosynthesis to CAM, with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues. CAM pathway genes were enriched with cis-regulatory elements associated with the regulation of circadian clock genes, providing the first cis-regulatory link between CAM and circadian clock regulation. Pineapple CAM photosynthesis evolved by the reconfiguration of pathways in C3 plants, through the regulatory neofunctionalization of preexisting genes and not through the acquisition of neofunctionalized genes via whole-genome or tandem gene duplication.

A new double right border binary vector for producing marker-free transgenic plants

PubMed Central

2013-01-01

Background Once a transgenic plant is developed, the selectable marker gene (SMG) becomes unnecessary in the plant. In fact, the continued presence of the SMG in the transgenic plant may cause unexpected pleiotropic effects as well as environmental or biosafety issues. Several methods for removal of SMGs that have been reported remain inaccessible due to protection by patents, while development of new ones is expensive and cost prohibitive. Here, we describe the development of a new vector for producing marker-free plants by simply adapting an ordinary binary vector to the double right border (DRB) vector design using conventional cloning procedures. Findings We developed the DRB vector pMarkfree5.0 by placing the bar gene (representing genes of interest) between two copies of T-DNA right border sequences. The β-glucuronidase (gus) and nptII genes (representing the selectable marker gene) were cloned next followed by one copy of the left border sequence. When tested in a model species (tobacco), this vector system enabled the generation of 55.6% kanamycin-resistant plants by Agrobacterium-mediated transformation. The frequency of cotransformation of the nptII and bar transgenes using the vector was 66.7%. Using the leaf bleach and Basta assays, we confirmed that the nptII and bar transgenes were coexpressed and segregated independently in the transgenic plants. This enable separation of the transgenes in plants cotransformed using pMarkfree5.0. Conclusions The results suggest that the DRB system developed here is a practical and effective approach for separation of gene(s) of interest from a SMG and production of SMG-free plants. Therefore this system could be instrumental in production of “clean” plants containing genes of agronomic importance. PMID:24207020

Handmade Cloned Transgenic Sheep Rich in Omega-3 Fatty Acids

PubMed Central

Dou, Hongwei; Chen, Lei; Chen, Longxin; Lin, Lin; Tan, Pingping; Vajta, Gabor; Gao, Jianfeng; Du, Yutao; Ma, Runlin Z.

2013-01-01

Technology of somatic cell nuclear transfer (SCNT) has been adapted worldwide to generate transgenic animals, although the traditional procedure relies largely on instrumental micromanipulation. In this study, we used the modified handmade cloning (HMC) established in cattle and pig to produce transgenic sheep with elevated levels of omega-3 (n−3) fatty acids. Codon-optimized nematode mfat-1 was inserted into a eukaryotic expression vector and was transferred into the genome of primary ovine fibroblast cells from a male Chinese merino sheep. Reverse transcriptase PCR, gas chromatography, and chromosome analyses were performed to select nuclear donor cells capable of converting omega-6 (n−6) into n−3 fatty acids. Blastocysts developed after 7 days of in vitro culture were surgically transplanted into the uterus of female ovine recipients of a local sheep breed in Xinjiang. For the HMC, approximately 8.9% (n  = 925) of reconstructed embryos developed to the blastocyst stage. Four recipients became pregnant after 53 blastocysts were transplanted into 29 naturally cycling females, and a total of 3 live transgenic lambs were produced. Detailed analyses on one of the transgenic lambs revealed a single integration of the modified nematode mfat-1 gene at sheep chromosome 5. The transgenic sheep expressed functional n−3 fatty acid desaturase, accompanied by more than 2-folds reduction of n−6/n−3 ratio in the muscle (p<0.01) and other major organs/tissues (p<0.05). To our knowledge, this is the first report of transgenic sheep produced by the HMC. Compared to the traditional SCNT method, HMC showed an equivalent efficiency but proved cheaper and easier in operation. PMID:23437077

Event-specific plasmid standards and real-time PCR methods for transgenic Bt11, Bt176, and GA21 maize and transgenic GT73 canola.

PubMed

Taverniers, Isabel; Windels, Pieter; Vaïtilingom, Marc; Milcamps, Anne; Van Bockstaele, Erik; Van den Eede, Guy; De Loose, Marc

2005-04-20

Since the 18th of April 2004, two new regulations, EC/1829/2003 on genetically modified food and feed products and EC/1830/2003 on traceability and labeling of GMOs, are in force in the EU. This new, comprehensive regulatory framework emphasizes the need of an adequate tracing system. Unique identifiers, such as the transgene genome junction region or a specific rearrangement within the transgene DNA, should form the basis of such a tracing system. In this study, we describe the development of event-specific tracing systems for transgenic maize lines Bt11, Bt176, and GA21 and for canola event GT73. Molecular characterization of the transgene loci enabled us to clone an event-specific sequence into a plasmid vector, to be used as a marker, and to develop line-specific primers. Primer specificity was tested through qualitative PCRs and dissociation curve analysis in SYBR Green I real-time PCRs. The primers were then combined with event-specific TaqMan probes in quantitative real-time PCRs. Calibration curves were set up both with genomic DNA samples and the newly synthesized plasmid DNA markers. It is shown that cloned plasmid GMO target sequences are perfectly suitable as unique identifiers and quantitative calibrators. Together with an event-specific primer pair and a highly specific TaqMan probe, the plasmid markers form crucial components of a unique and straighforward tracing system for Bt11, Bt176, and GA21 maize and GT73 canola events.

Genome-wide organization and expression profiling of the R2R3-MYB transcription factor family in pineapple (Ananas comosus).

PubMed

Liu, Chaoyang; Xie, Tao; Chen, Chenjie; Luan, Aiping; Long, Jianmei; Li, Chuhao; Ding, Yaqi; He, Yehua

2017-07-01

The MYB proteins comprise one of the largest families of plant transcription factors, which are involved in various plant physiological and biochemical processes. Pineapple (Ananas comosus) is one of three most important tropical fruits worldwide. The completion of pineapple genome sequencing provides a great opportunity to investigate the organization and evolutionary traits of pineapple MYB genes at the genome-wide level. In the present study, a total of 94 pineapple R2R3-MYB genes were identified and further phylogenetically classified into 26 subfamilies, as supported by the conserved gene structures and motif composition. Collinearity analysis indicated that the segmental duplication events played a crucial role in the expansion of pineapple MYB gene family. Further comparative phylogenetic analysis suggested that there have been functional divergences of MYB gene family during plant evolution. RNA-seq data from different tissues and developmental stages revealed distinct temporal and spatial expression profiles of the AcMYB genes. Further quantitative expression analysis showed the specific expression patterns of the selected putative stress-related AcMYB genes in response to distinct abiotic stress and hormonal treatments. The comprehensive expression analysis of the pineapple MYB genes, especially the tissue-preferential and stress-responsive genes, could provide valuable clues for further function characterization. In this work, we systematically identified AcMYB genes by analyzing the pineapple genome sequence using a set of bioinformatics approaches. Our findings provide a global insight into the organization, phylogeny and expression patterns of the pineapple R2R3-MYB genes, and hence contribute to the greater understanding of their biological roles in pineapple.

Genetically engineered hairy root cultures of Hyoscyamus senecionis and H. muticus: ploidy as a promising parameter in the metabolic engineering of tropane alkaloids.

PubMed

Dehghan, Esmaeil; Reed, Darwin W; Covello, Patrick S; Hasanpour, Zeinab; Palazon, Javier; Oksman-Caldentey, Kirsi-Marja; Ahmadi, Farajollah Shahriari

2017-10-01

Tetraploidy improves overexpression of h6h and scopolamine production of H. muticus, while in H. senecionis, pmt overexpression and elicitation can be used as effective methods for increasing tropane alkaloids. The effects of metabolic engineering in a polyploid context were studied by overexpression of h6h in the tetraploid hairy root cultures of H. muticus. Flow cytometry analysis indicated genetic stability in the majority of the clones, while only a few clones showed genetic instability. Among all the diploid and tetraploid clones, the highest level of h6h transgene expression and scopolamine accumulation was interestingly observed in the tetraploid clones of H. muticus. Therefore, metabolic engineering of the tropane biosynthetic pathway in polyploids is suggested as a potential system for increasing the production of tropane alkaloids. Transgenic hairy root cultures of Hyoscyamus senecionis were also established. While overexpression of pmt in H. senecionis was correlated with a sharp increase in hyoscyamine production, the h6h-overexpressing clones were not able to accumulate higher levels of scopolamine than the leaves of intact plants. Applying methyl jasmonate was followed by a sharp increase in the expression of pmt and a drop in the expression of tropinone reductase II (trII) which consequently resulted in the higher biosynthesis of hyoscyamine and total alkaloids in H. senecionis.

Citrus and Prunuscopia-like retrotransposons.

PubMed

Asíns, M J; Monforte, A J; Mestre, P F; Carbonell, E A

1999-08-01

Many of the world's most important citrus cultivars ("Washington Navel", satsumas, clementines) have arisen through somatic mutation. This phenomenon occurs fairly often in the various species and varieties of the genus.The presence of copia-like retrotransposons has been investigated in fruit trees, especially citrus, by using a PCR assay designed to detect copia-like reverse transcriptase (RT) sequences. Amplification products from a genotype of each the following species Citrus sinensis, Citrus grandis, Citrus clementina, Prunus armeniaca and Prunus amygdalus, were cloned and some of them sequenced. Southern-blot hybridization using RT clones as probes showed that multiple copies are integrated throughout the citrus genome, while only 1-3 copies are detected in the P. armeniaca genome, which is in accordance with the Citrus and Prunus genome sizes. Sequence analysis of RT clones allowed a search for homologous sequences within three gene banks. The most similar ones correspond to RT domains of copia-like retrotransposons from unrelated plant species. Cluster analysis of these sequences has shown a great heterogeneity among RT domains cloned from the same genotype. This finding supports the hypothesis that horizontal transmission of retrotransposons has occurred in the past. The species presenting a RT sequence most similar to citrus RT clones is Gnetum montanum, a gymnosperm whose distribution area coincides with two of the main centers of origin of Citrus spp. A new C-methylated restriction DNA fragment containing a RT sequence is present in navel sweet oranges, but not in Valencia oranges from which the former originated suggesting, that retrotransposon activity might be, at least in part, involved in the genetic variability among sweet orange cultivars. Given that retrotransposons are quite abundant throughout the citrus genome, their activity should be investigated thoroughly before commercializing any transgenic citrus plant where the transgene(s) is part of a viral genome in order to avoid its possible recombination with an active retroelement. Focusing on other strategies to control virus diseases is recommended in citrus.

Functional and evolution characterization of SWEET sugar transporters in Ananas comosus.

PubMed

Guo, Chengying; Li, Huayang; Xia, Xinyao; Liu, Xiuyuan; Yang, Long

2018-02-05

Sugars will eventually be exported transporters (SWEETs) are a group of recently identified sugar transporters in plants that play important roles in diverse physiological processes. However, currently, limited information about this gene family is available in pineapple (Ananas comosus). The availability of the recently released pineapple genome sequence provides the opportunity to identify SWEET genes in a Bromeliaceae family member at the genome level. In this study, 39 pineapple SWEET genes were identified in two pineapple cultivars (18 AnfSWEET and 21 AnmSWEET) and further phylogenetically classified into five clades. A phylogenetic analysis revealed distinct evolutionary paths for the SWEET genes of the two pineapple cultivars. The MD2 cultivar might have experienced a different expansion than the F153 cultivar because two additional duplications exist, which separately gave rise to clades III and IV. A gene exon/intron structure analysis showed that the pineapple SWEET genes contained highly conserved exon/intron numbers. An analysis of public RNA-seq data and expression profiling showed that SWEET genes may be involved in fruit development and ripening processes. AnmSWEET5 and AnmSWEET11 were highly expressed in the early stages of pineapple fruit development and then decreased. The study increases the understanding of the roles of SWEET genes in pineapple. Copyright © 2018 Elsevier Inc. All rights reserved.

The draft genome of MD-2 pineapple using hybrid error correction of long reads

PubMed Central

Redwan, Raimi M.; Saidin, Akzam; Kumar, S. Vijay

2016-01-01

The introduction of the elite pineapple variety, MD-2, has caused a significant market shift in the pineapple industry. Better productivity, overall increased in fruit quality and taste, resilience to chilled storage and resistance to internal browning are among the key advantages of the MD-2 as compared with its previous predecessor, the Smooth Cayenne. Here, we present the genome sequence of the MD-2 pineapple (Ananas comosus (L.) Merr.) by using the hybrid sequencing technology from two highly reputable platforms, i.e. the PacBio long sequencing reads and the accurate Illumina short reads. Our draft genome achieved 99.6% genome coverage with 27,017 predicted protein-coding genes while 45.21% of the genome was identified as repetitive elements. Furthermore, differential expression of ripening RNASeq library of pineapple fruits revealed ethylene-related transcripts, believed to be involved in regulating the process of non-climacteric pineapple fruit ripening. The MD-2 pineapple draft genome serves as an example of how a complex heterozygous genome is amenable to whole genome sequencing by using a hybrid technology that is both economical and accurate. The genome will make genomic applications more feasible as a medium to understand complex biological processes specific to pineapple. PMID:27374615

A comparison of tensile properties of polyester composites reinforced with pineapple leaf fiber and pineapple peduncle fiber

NASA Astrophysics Data System (ADS)

Juraidi, J. M.; Shuhairul, N.; Syed Azuan, S. A.; Intan Saffinaz Anuar, Noor

2013-12-01

Pineapple fiber which is rich in cellulose, relatively inexpensive, and abundantly available has the potential for polymer reinforcement. This research presents a study of the tensile properties of pineapple leaf fiber and pineapple peduncle fiber reinforced polyester composites. Composites were fabricated using leaf fiber and peduncle fiber with varying fiber length and fiber loading. Both fibers were mixed with polyester composites the various fiber volume fractions of 4, 8 and 12% and with three different fiber lengths of 10, 20 and 30 mm. The composites panels were fabricated using hand lay-out technique. The tensile test was carried out in accordance to ASTM D638. The result showed that pineapple peduncle fiber with 4% fiber volume fraction and fiber length of 30 mm give highest tensile properties. From the overall results, pineapple peduncle fiber shown the higher tensile properties compared to pineapple leaf fiber. It is found that by increasing the fiber volume fraction the tensile properties has significantly decreased but by increasing the fiber length, the tensile properties will be increased proportionally. Minitab software is used to perform the two-way ANOVA analysis to measure the significant. From the analysis done, there is a significant effect of fiber volume fraction and fiber length on the tensile properties.

Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma.

PubMed

Ozawa, Masayuki; Himaki, Takehiro; Ookutsu, Shoji; Mizobe, Yamato; Ogawa, Junki; Miyoshi, Kazuchika; Yabuki, Akira; Fan, Jianglin; Yoshida, Mitsutoshi

2015-01-01

High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

The pineapple genome and the evolution of CAM photosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ming, Ray; VanBuren, Robert; Wai, Ching Man

Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water-use efficiency, and the second most important tropical fruit. We sequenced the genomes of pineapple varieties F153 and MD2 and a wild pineapple relative, Ananas bracteatus accession CB5. The pineapple genome has one fewer ancient whole-genome duplication event than sequenced grass genomes and a conserved karyotype with seven chromosomes from before the ρ duplication event. The pineapple lineage has transitioned from C 3 photosynthesis to CAM, with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues.more » CAM pathway genes were enriched with cis-regulatory elements associated with the regulation of circadian clock genes, providing the first cis-regulatory link between CAM and circadian clock regulation. Lastly, we found pineapple CAM photosynthesis evolved by the reconfiguration of pathways in C 3 plants, through the regulatory neofunctionalization of preexisting genes and not through the acquisition of neofunctionalized genes via whole-genome or tandem gene duplication.« less

Aroma Volatile Compounds from Two Fresh Pineapple Varieties in China

PubMed Central

Zheng, Liang-Yong; Sun, Guang-Ming; Liu, Yu-Ge; Lv, Ling-Ling; Yang, Wen-Xiu; Zhao, Wei-Feng; Wei, Chang-Bin

2012-01-01

Volatile compounds from two pineapples varieties (Tainong No.4 and No.6) were isolated by headspace solid phase microextraction (HS-SPME) and identified and quantified by gas chromatography-mass spectrometry (GC/MS). In the Tainong No. 4 and No. 6 pineapples, a total of 11 and 28 volatile compounds were identified according to their retention time on capillary columns and their mass spectra, and quantified with total concentrations of 1080.44 μg·kg−1 and 380.66 μg·kg−1 in the Tainong No.4 and No. 6 pineapples, respectively. The odor active values (OAVs) of volatile compounds from pineapples were also calculated. According to the OAVs, four compounds were defined as the characteristic aroma compounds for the Tainong No. 4 pineapple, including furaneol, 3-(methylthio)propanoic acid methyl ester, 3-(methylthio)propanoic acid ethyl ester and δ-octalactone. The OAVs of five compounds including ethyl-2-methylbutyrate, methyl-2-methylbutyrate, 3-(methylthio)propanoic acid ethyl ester, ethyl hexanoate and decanal were considered to be the characteristic aroma compounds for the Tainong No. 6 pineapple. PMID:22837701

Application of Pineapple Juice in the Fish Digestion Process for Carcinogenic Liver Fluke Metacercaria Collection

PubMed Central

Sripan, Panupan; Aukkanimart, Ratchadawan; Boonmars, Thidarut; Pranee, Sriraj; Songsri, Jiraporn; Boueroy, Parichart; Khueangchaingkhwang, Sukhonthip; Pumhirunroj, Benjamabhorn; Artchayasawat, Atchara

2017-01-01

Pepsin is common digestive enzyme used for fish digestion in the laboratory to collect trematode metacercariae. In a field study, to survey the infected fish is needed a huge yield of pepsin and it is very expensive. Therefore, our purpose of this study was to investigate the candidate enzyme from pineapple juice which has a digestive enzyme called bromelain, a mixture of proteolytic enzymes, to digest fish in order to harvest metacercariae. Fish were divided into 2 groups: one group in which metacercariae were harvested using acid pepsin as a control and other groups in which the fish was digested using fresh pineapple juices. The results showed that pineapple juice is able to digest fish similarly to pepsin. The Pattavia pineapple juice had the highest number of metacercariae similar to the control. For Trat Si Thong pineapple juice, we found the number of metacercariae was less than control. This result suggests that the Pattavia pineapple juice was optimal juice for fish digestion to metacercaria collection and can be used instread of pepsin acid. PMID:28441786

The pineapple genome and the evolution of CAM photosynthesis

DOE PAGES

Ming, Ray; VanBuren, Robert; Wai, Ching Man; ...

2015-11-02

Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water-use efficiency, and the second most important tropical fruit. We sequenced the genomes of pineapple varieties F153 and MD2 and a wild pineapple relative, Ananas bracteatus accession CB5. The pineapple genome has one fewer ancient whole-genome duplication event than sequenced grass genomes and a conserved karyotype with seven chromosomes from before the ρ duplication event. The pineapple lineage has transitioned from C 3 photosynthesis to CAM, with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues.more » CAM pathway genes were enriched with cis-regulatory elements associated with the regulation of circadian clock genes, providing the first cis-regulatory link between CAM and circadian clock regulation. Lastly, we found pineapple CAM photosynthesis evolved by the reconfiguration of pathways in C 3 plants, through the regulatory neofunctionalization of preexisting genes and not through the acquisition of neofunctionalized genes via whole-genome or tandem gene duplication.« less

Plant fatty acid hydroxylases

DOEpatents

Somerville, Chris; Broun, Pierre; van de Loo, Frank

2001-01-01

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

Cloning of two individual cDNAS encoding 9-cis-epoxycarotenoid dioxygenase from Gentiana lutea, their tissue-specific expression and physiological effect in transgenic tobacco.

PubMed

Zhu, Changfu; Kauder, Friedrich; Römer, Susanne; Sandmann, Gerhard

2007-02-01

Two 9-cis-epoxycarotenoid dioxygenase (NCED) cDNAs have been cloned from a petal library of Gentiana lutea. Both cDNAs carry a putative transit sequence for chloroplast import and differ mainly in their length and the 5'-flanking regions. GlNCED1 was evolutionary closely related to Arabidopsis thaliana NCED6 whereas GlNCED2 showed highest homology to tomato NCED1 and A. thaliana NCED3. The amounts of GlNCED2 transcript were below Northern detection in G. lutea. In contrast, GlNCED1 was specifically expressed at higher levels in developing flowers when petals start appearing. By genetic engineering of tobacco with coding regions of either gene under a constitutive promoter, their function was further analyzed. Although mRNA of both genes was detectable in the corresponding transgenic plants, a physiological effect was only found for GlNCED1 but not for GlNCED2. In germination experiments of GlNCED1 transgenic lines, delayed radicle formation and cotyledon appearance were observed. However, the transformants exhibited no improved tolerance against desiccation stress. In contrast to other plants with over-expressed NCEDs, prolonged delay of seed germination is the only abscisic-acid-related phenotypic effect in the GlNCED1 transgenic lines.

Effectiveness of radiation processing for elimination of Salmonella Typhimurium from minimally processed pineapple (Ananas comosus Merr.).

PubMed

Shashidhar, Ravindranath; Dhokane, Varsha S; Hajare, Sachin N; Sharma, Arun; Bandekar, Jayant R

2007-04-01

The microbiological quality of market samples of minimally processed (MP) pineapple was examined. The effectiveness of radiation treatment in eliminating Salmonella Typhimurium from laboratory inoculated ready-to-eat pineapple slices was also studied. Microbiological quality of minimally processed pineapple samples from Mumbai market was poor; 8.8% of the samples were positive for Salmonella. D(10) (the radiation dose required to reduce bacterial population by 90%) value for S. Typhimurium inoculated in pineapple was 0.242 kGy. Inoculated pack studies in minimally processed pineapple showed that the treatment with a 2-kGy dose of gamma radiation could eliminate 5 log CFU/g of S. Typhimurium. The pathogen was not detected from radiation-processed samples up to 12 d during storage at 4 and 10 degrees C. The processing of market samples with 1 and 2 kGy was effective in improving the microbiological quality of these products.

Effects of elevated temperature postharvest on color aspect, physiochemical characteristics, and aroma components of pineapple fruits.

PubMed

Liu, Chuanhe; Liu, Yan

2014-12-01

In this work, 2 separate experiments were performed to describe the influence of elevated temperature treatments postharvest on the color, physiochemical characteristics and aroma components of pineapple fruits during low-temperature seasons. The L* (lightness) values of the skin and pulp of pineapple fruits were decreased. The a* (greenness-redness) and b* (blueness-yellowness) values of the skin and pulp were all markedly increased. The elevated temperature significantly increased the contents of total soluble solids (TSS) and slightly affected contents of vitamin C (nonsignificant). Titratable acidity (TA) of pineapple fruits were notably decreased, whereas the values of TSS/TA of pineapple fruits were significantly increased. The firmness of the pineapple fruits decreased and more esters and alkenes were identified. The total relative contents of esters were increased, and the total relative contents of alkenes were decreased. © 2014 Institute of Food Technologists®

Genetically engineered livestock for biomedical models.

PubMed

Rogers, Christopher S

2016-06-01

To commemorate Transgenic Animal Research Conference X, this review summarizes the recent progress in developing genetically engineered livestock species as biomedical models. The first of these conferences was held in 1997, which turned out to be a watershed year for the field, with two significant events occurring. One was the publication of the first transgenic livestock animal disease model, a pig with retinitis pigmentosa. Before that, the use of livestock species in biomedical research had been limited to wild-type animals or disease models that had been induced or were naturally occurring. The second event was the report of Dolly, a cloned sheep produced by somatic cell nuclear transfer. Cloning subsequently became an essential part of the process for most of the models developed in the last 18 years and is stilled used prominently today. This review is intended to highlight the biomedical modeling achievements that followed those key events, many of which were first reported at one of the previous nine Transgenic Animal Research Conferences. Also discussed are the practical challenges of utilizing livestock disease models now that the technical hurdles of model development have been largely overcome.

Creating genetically modified pigs by using nuclear transfer

PubMed Central

Lai, Liangxue; Prather, Randall S

2003-01-01

Nuclear transfer (NT) is a procedure by which genetically identical individuals can be created. The technology of pig somatic NT, including in vitro maturation of oocytes, isolation and treatment of donor cells, artificial activation of reconstructed oocytes, embryo culture and embryo transfer, has been intensively studied in recent years, resulting in birth of cloned pigs in many labs. While it provides an efficient method for producing transgenic pigs, more importantly, it is the only way to produce gene-targeted pigs. So far pig cloning has been successfully used to produce transgenic pigs expressing the green fluorescence protein, expand transgenic pig groups and create gene targeted pigs which are deficient of alpha-1,3-galactosyltransferase. The production of pigs with genetic modification by NT is now in the transition from investigation to practical use. Although the efficiency of somatic cell NT in pig, when measured as development to term as a proportion of oocytes used, is not high, it is anticipated that the ability of making specific modifications to the swine genome will result in this technology having a large impact not only on medicine but also on agriculture. PMID:14613542

Cognitive enhancing of pineapple extract and juice in scopolamine-induced amnesia in mice

PubMed Central

Momtazi-borojeni, Amir Abbas; Sadeghi-Aliabadi, Hojjat; Rabbani, Mohammed; Ghannadi, Alireza; Abdollahi, Elham

2017-01-01

The objective of the present study was to evaluate the cognitive enhancing of pineapple juice and ethanolic extract in scopolamine-induced cognitive deficit mice. The ethanolic extract of pineapple (Ananas comosus (L.) Merr.) was prepared by maceration method and its juice was obtained by a homogenizer. Object recognition task was used to evaluate the mice memory. Exploration time in the first and second trial was recorded. The differences in exploration time between a familiar and a novel object in the second trial were taken as a memory index. Animals were randomly assigned into 15 groups of 6 each including: control group (normal saline + vehicle), positive control group (scopolamine + rivastigmine), seven experimental groups (received scopolamine alone or scopolamine + ethanolic extract of pineapple in different doses), six other experimental groups were treated by ethanolic extract or juice of pineapple in different doses. Scopolamine (100 μL, 1 mg/kg, i.p.) and pineapple juice or extract (50, 75 and 100 mg/kg, i.p.) were administered 40 and 30 min before starting the second trial in the experimental groups. Object discrimination was impaired after scopolamine administration. Results showed that juice and ethanolic extract of pineapple significantly restored object recognition ability in mice treated with scopolamine. These finding suggested that pineapple had a protective role against scopolamine-induced amnesia, indicating its ability in management of cognitive disorders. PMID:28626484

Germ line transmission of a yeast artificial chromosome spanning the murine [alpha][sub 1](I) collagen locus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauss, W.M.; Dausman, J.; Beard, C.

Molecular complementation of mutant phenotypes by transgenic technology is a potentially important tool for gene identification. A technology was developed to allow the transfer of a physically intact yeast artificial chromosome (YAC) into the germ line of the mouse. A purified 150-kilobase YAC encompassing the murine gene Col1a1 was efficiently introduced into embryonic stem (ES) cells via lipofection. Chimeric founder mice were derived from two transfected ES cell clones. These chimeras transmitted the full length transgene through the germ line, generating two transgenic mouse strains. Transgene expression was visualized as nascent transcripts in interphase nuclei and quantitated by ribonuclease protectionmore » analysis. Both assays indicated that the transgene was expressed at levels comparable to the endogenous collagen gene. 32 refs., 3 figs., 1 tab.« less

Fresh-Cut Pineapple as a New Carrier of Probiotic Lactic Acid Bacteria

PubMed Central

Russo, Pasquale; de Chiara, Maria Lucia Valeria; Vernile, Anna; Amodio, Maria Luisa; Arena, Mattia Pia; Capozzi, Vittorio; Massa, Salvatore; Spano, Giuseppe

2014-01-01

Due to the increasing interest for healthy foods, the feasibility of using fresh-cut fruits to vehicle probiotic microorganisms is arising scientific interest. With this aim, the survival of probiotic lactic acid bacteria, belonging to Lactobacillus plantarum and Lactobacillus fermentum species, was monitored on artificially inoculated pineapple pieces throughout storage. The main nutritional, physicochemical, and sensorial parameters of minimally processed pineapples were monitored. Finally, probiotic Lactobacillus were further investigated for their antagonistic effect against Listeria monocytogenes and Escherichia coli O157:H7 on pineapple plugs. Our results show that at eight days of storage, the concentration of L. plantarum and L. fermentum on pineapples pieces ranged between 7.3 and 6.3 log cfu g−1, respectively, without affecting the final quality of the fresh-cut pineapple. The antagonistic assays indicated that L. plantarum was able to inhibit the growth of both pathogens, while L. fermentum was effective only against L. monocytogenes. This study suggests that both L. plantarum and L. fermentum could be successfully applied during processing of fresh-cut pineapples, contributing at the same time to inducing a protective effect against relevant foodborne pathogens. PMID:25093163

Studies into the phenolic patterns of different tissues of pineapple (Ananas comosus [L.] Merr.) infructescence by HPLC-DAD-ESI-MS (n) and GC-MS analysis.

PubMed

Steingass, Christof B; Glock, Mona P; Schweiggert, Ralf M; Carle, Reinhold

2015-08-01

In a comprehensive study, more than 60 phenolic compounds were detected in methanolic extracts from different tissues of pineapple infructescence by high-performance liquid chromatography with diode array detection and electrospray ionisation multiple-stage mass spectrometry (HPLC-DAD-ESI-MS (n) ) as well as by gas chromatography-mass spectrometry (GC-MS). The analytical workflow combining both methods revealed numerous compounds assigned for the first time as pineapple constituents by their mass fragmentations. Pineapple crown tissue was characterised by depsides of p-coumaric and ferulic acid. In contrast, major phenolic compounds in pineapple pulp extracts were assigned to diverse S-p-coumaryl, S-coniferyl and S-sinapyl derivatives of glutathione, N-L-γ-glutamyl-L-cysteine and L-cysteine, which were also identified in the peel. The latter was additionally characterised by elevated concentrations of p-coumaric, ferulic and caffeic acid depsides and glycerides, respectively. Two peel-specific cyanidin hexosides were found. Elevated concentrations of isomeric N,N'-diferuloylspermidines may be a useful tool for the detection of fraudulent peel usage for pineapple juice production. Mass fragmentation pathways of characteristic pineapple constituents are proposed, and their putative biological functions are discussed.

A review of oral vaccination with transgenic vegetables.

PubMed

Tacket, C O; Mason, H S

1999-08-01

Mucosal immunization of the gastrointestinal tract is an effective way to stimulate local and systemic immune responses. Oral vaccines must be formulated in such a way that antigens are protected as they pass through the adverse environment of the stomach and are delivered to the mucosal inductive sites. Vaccine antigens cloned into edible transgenic plants are a promising new delivery system for oral vaccines. Such vaccines could be safe, inexpensive, and multicomponent.

Vascular endothelium-specific overexpression of human catalase in cloned pigs

PubMed Central

Samuel, M.; Mahan, E.; Padilla, J.; Simmons, G. H.; Arce-Esquivel, A. A.; Bender, S. B.; Whitworth, K. M.; Hao, Y. H.; Murphy, C. N.; Walters, E. M.; Prather, R. S.; Laughlin, M. H.

2012-01-01

The objective of this study was to develop transgenic Yucatan minipigs that overexpress human catalase (hCat) in an endothelial-specific manner. Catalase metabolizes hydrogen peroxide (H2O2), an important regulator of vascular tone that contributes to diseases such as atherosclerosis and preeclampsia. A large animal model to study reduced endothelium-derived H2O2 would therefore generate valuable translational data on vascular regulation in health and disease. Yucatan minipig fetal fibroblasts stably co-transfected with human catalase (Tie2-hCat) and eGFP expression constructs were isolated into single-cell populations. The presence of the Tie2-hCat transgene in individual colonies of fibroblasts was determined by PCR. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were transferred per surrogate sow (n = 4). All four surrogates maintained pregnancies and piglets were delivered by cesarean section. Nine male piglets from three of the four litters carried the Tie2-hCat transgene. Expression of human catalase mRNA and overall elevated catalase protein in isolated umbilical endothelial cells from transgenic piglets were verified by RT–PCR and western blot, respectively, and endothelial localization was confirmed by immunohistochemistry. Increased enzymatic activity of catalase in transgenic versus wild-type endothelial cells was inferred based on significantly reduced levels of H2O2 in culture. The similarities in swine and human cardiovascular anatomy and physiology will make this pig model a valuable source of information on the putative role of endothelium-derived H2O2 in vasodilation and in the mechanisms underlying vascular health and disease. PMID:21170678

Overexpression of LEAFY in apple leads to a columnar phenotype with shorter internodes.

PubMed

Flachowsky, Henryk; Hättasch, Conny; Höfer, Monika; Peil, Andreas; Hanke, Magda-Viola

2010-01-01

To break the juvenile stage of apple (Malus x domestica Borkh.) we transferred the LFY gene of Arabidopsis into the genome of the apple cv. 'Pinova'. A total of five transgenic clones constitutively overexpressing the LFY gene were obtained. Approximately, 20 shoots of each clone were rooted and transferred to the glasshouse. No flowers were obtained on transgenic plants during the first 2 years of cultivation. Evaluation of the expression of possible LFY targets revealed that no transcripts could be detected for MdAP1-1 and MdAP1-2. MdTFL1 was unaffected. Based on the absence of the LFY core-binding sequence within promoter sequences of MdAP1-1 and MdAP1-2, it was concluded that LFY was not able to induce these genes. The LFY genes of apple were unaffected in transgenic plants and sequence alignments of the C-terminal amino acid sequence showed a high conservation of these proteins. A change in binding ability to DNA can therefore be excluded. Instead of early flowering, the transgenic plants showed an altered phenotype, which is similar to the columnar phenotype of the 'McIntosh Wijcik' mutant of apple. The transgenic plants showed shortened internodes and a significantly reduced length of the regrowing shoot. A negative correlation was observed between the length of the regrowing shoot and the LFY mRNA transcript level. Furthermore, the LFY transgenic apple plants showed an increased shoot diameter at node 20, which was positively correlated with the LFY mRNA transcript level. Based on our results, we assume an alternative role of LFY in apple.

Generation of selectable marker-free transgenic eggplant resistant to Alternaria solani using the R/RS site-specific recombination system.

PubMed

Darwish, Nader Ahmed; Khan, Raham Sher; Ntui, Valentine Otang; Nakamura, Ikuo; Mii, Masahiro

2014-03-01

Marker-free transgenic eggplants, exhibiting enhanced resistance to Alternaria solani , can be generated on plant growth regulators (PGRs)- and antibiotic-free MS medium employing the multi-auto-transformation (MAT) vector, pMAT21 - wasabi defensin , wherein isopentenyl transferase ( ipt ) gene is used as a positive selection marker. Use of the selection marker genes conferring antibiotic or herbicide resistance in transgenic plants has been considered a serious problem for environment and the public. Multi-auto-transformation (MAT) vector system has been one of the tools to excise the selection marker gene and produce marker-free transgenic plants. Ipt gene was used as a selection marker gene. Wasabi defensin gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), was used as a gene of interest. Wasabi defensin gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledon explants of eggplant were cultured on PGRs- and antibiotic-free MS medium. Extreme shooty phenotype/ipt shoots were produced by the explants infected with the pMAT21-wasabi defensin (WD). The same PGRs- and antibiotic-free MS medium was used in subcultures of the ipt shoots. Subsequently, morphologically normal shoots emerged from the Ipt shoots. Molecular analyses of genomic DNA from transgenic plants confirmed the integration of the WD gene and excision of the selection marker (ipt gene). Expression of the WD gene was confirmed by RT-PCR and Northern blot analyses. In vitro whole plant and detached leaf assay of the marker-free transgenic plants exhibited enhanced resistance against Alternaria solani.

The pineapple genome and the evolution of CAM photosynthesis

USDA-ARS?s Scientific Manuscript database

Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water-use efficiency, and the second most important tropical fruit. We sequenced the genomes of pineapple varieties F153 ...

From Wine to Pineapples: Delta Company Takes on New Satellite Role

DTIC Science & Technology

2011-01-01

2011 Spring / Summer edition army Space Journal 2F Delta Company Takes on New Satellite Role FROM Wine TO PineAPPLeS WAHIAWA, Hawaii – 2011 has yet...next to pineapple fields on the island of Oahu, Hawaii. Delta Company has provided network and payload control for the Defense Satellite...1. REPORT DATE 2011 2. REPORT TYPE 3. DATES COVERED 00-00-2011 to 00-00-2011 4. TITLE AND SUBTITLE From Wine to Pineapples : Delta Company

Discovery of precursor and mature microRNAs and their putative gene targets using high-throughput sequencing in pineapple (Ananas comosus var. comosus).

PubMed

Yusuf, Noor Hydayaty Md; Ong, Wen Dee; Redwan, Raimi Mohamed; Latip, Mariam Abd; Kumar, S Vijay

2015-10-15

MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that negatively regulate gene expression, resulting in the silencing of target mRNA transcripts through mRNA cleavage or translational inhibition. MiRNAs play significant roles in various biological and physiological processes in plants. However, the miRNA-mediated gene regulatory network in pineapple, the model tropical non-climacteric fruit, remains largely unexplored. Here, we report a complete list of pineapple mature miRNAs obtained from high-throughput small RNA sequencing and precursor miRNAs (pre-miRNAs) obtained from ESTs. Two small RNA libraries were constructed from pineapple fruits and leaves, respectively, using Illumina's Solexa technology. Sequence similarity analysis using miRBase revealed 579,179 reads homologous to 153 miRNAs from 41 miRNA families. In addition, a pineapple fruit transcriptome library consisting of approximately 30,000 EST contigs constructed using Solexa sequencing was used for the discovery of pre-miRNAs. In all, four pre-miRNAs were identified (MIR156, MIR399, MIR444 and MIR2673). Furthermore, the same pineapple transcriptome was used to dissect the function of the miRNAs in pineapple by predicting their putative targets in conjunction with their regulatory networks. In total, 23 metabolic pathways were found to be regulated by miRNAs in pineapple. The use of high-throughput sequencing in pineapples to unveil the presence of miRNAs and their regulatory pathways provides insight into the repertoire of miRNA regulation used exclusively in this non-climacteric model plant. Copyright © 2015 Elsevier B.V. All rights reserved.

Influence of modified atmosphere packaging on volatile compounds and physicochemical and antioxidant attributes of fresh-cut pineapple (Ananas comosus).

PubMed

Montero-Calderón, Marta; Rojas-Graü, María Alejandra; Aguiló-Aguayo, Ingrid; Soliva-Fortuny, Robert; Martín-Belloso, Olga

2010-04-28

The effects of modified atmosphere packaging on volatile compound content and physicochemical and antioxidant attributes of Gold cultivar fresh-cut pineapples were assessed throughout storage at 5 degrees C. Fresh-cut pineapple pieces were packed under LO (low oxygen, 12% O(2), 1% CO(2)), AIR (20.9% O(2)) and HO (high oxygen, 38% O(2)) headspace atmospheres. Methyl butanoate, methyl 2-methylbutanoate, and methyl hexanoate were the most abundant volatiles regardless of the packaging atmosphere and days of storage; whereas most odor active volatiles were methyl and ethyl 2-methylbutanoate, 2,5-dimethyl-4-methoxy-3(2H)-furanone and ethyl hexanoate. Physicochemical attributes of pineapple did not significantly vary, whereas vitamin C content and total antioxidant capacity were lower for fresh-cut pineapple in HO (488 +/- 38 mg/100 mg(fw) and 54.4 +/- 5.7%, respectively) than for LO and AIR packages. Storage life of fresh-cut pineapple was limited to 14 days by volatile compounds losses and fermentation processes.

Future and applications of cloning.

PubMed

Trounson, Alan O

2006-01-01

The birth of viable offspring from somatic cell nuclear transfer (SCNT) in mammals caused a major re-examination of the understanding of the commitment of cells to specific tissue lineages during differentiation. The questions of whether cells undergo dedifferentiation or transdifferentiation during the development of offspring and how these changes are controlled is a source of ongoing debate that is yet to be resolved. Irrespective of the outcome of this debate, it is clear that cloning using SCNT has a place and purpose in the future of research and animal breeding. The future uses of SCNT could include the production of transgenic mice, the production of transgenic livestock and assisting with the re-establishment of endangered species. Human medicine also would benefit from future use of SCNT because it would allow the production of patient-specific embryonic stem cells.

Functional characterization of recombinant bromelain of Ananas comosus expressed in a prokaryotic system.

PubMed

George, Susan; Bhasker, Salini; Madhav, Harish; Nair, Archana; Chinnamma, Mohankumar

2014-02-01

Bromelain (BRM) is a defense protein present in the fruit and stem of pineapple (Ananas comosus) and it is grouped as a cysteine protease enzyme with diversified medicinal uses. Based on its therapeutic applications, bromelain has got sufficient attention in pharmaceutical industries. In the present study, the full coding gene of bromelain in pineapple stem (1,093 bp) was amplified by RT-PCR. The PCR product was cloned, sequenced, and characterized. The sequence analysis of the gene revealed the single nucleotide polymorphism and its phylogenetic relatedness. The peptide sequence deduced from the gene showed the amino acid variations, physicochemical properties and secondary and tertiary structural features of the protein. The full BRM gene was transformed to prokaryotic vector pET32b and expressed in Escherichia coli BL21 DE3pLysS host cells successfully. The identity of the recombinant bromelain (rBRM) protein was confirmed by Western blot analysis using anti-BRM-rabbit IgG antibody. The activity of recombinant bromelain compared with purified native bromelain was determined by protease assay. The inhibitory effect of rBRM compared with native BRM in the growth of Gram-positive and Gram-negative strains of Streptococcus agalactiae and Escherichia coli O111 was evident from the antibacterial sensitivity test. To the best of our knowledge, this is the first report showing the bactericidal property of rBRM expressed in a prokaryotic system.

Over-expression of 3-hydroxy-3- methylglutaryl-coenzyme A reductase 1 (hmgr1) gene under super-promoter for enhanced latex biosynthesis in rubber tree (Hevea brasiliensis Muell. Arg.).

PubMed

Jayashree, R; Nazeem, P A; Rekha, K; Sreelatha, S; Thulaseedharan, A; Krishnakumar, R; Kala, R G; Vineetha, M; Leda, P; Jinu, U; Venkatachalam, P

2018-06-01

Natural rubber (cis-1, 4-polyisoprene) is being produced from bark laticifer cells of Hevea brasiliensis and the popular high latex yielding Indian rubber clones are easily prone to onset of tapping panel dryness syndrome (TPD) which is considered as a physiological syndrome affecting latex production either partially or completely. This report describes an efficient protocol for development of transgenic rubber plants by over-expression of 3-hydroxy 3-methylglutaryl Co-enzyme A reductase 1 (hmgr1) gene which is considered as rate limiting factor for latex biosynthesis via Agrobacterium-mediated transformation. The pBIB plasmid vector containing hmgr1 gene cloned under the control of a super-promoter was used for genetic transformation using embryogenic callus. Putatively transgenic cell lines were obtained on selection medium and produced plantlets with 44% regeneration efficiency. Transgene integration was confirmed by PCR amplification of 1.8 kb hmgr1 and 0.6 kb hpt genes from all putatively transformed callus lines as well as transgenic plants. Southern blot analysis showed the stable integration and presence of transgene in the transgenic plants. Over expression of hmgr1 transgene was determined by Northern blot hybridization, semi-quantitative PCR and real-time PCR (qRT-PCR) analysis. Accumulation of hmgr1 mRNA transcripts was more abundant in transgenic plants than control. Increased level of photosynthetic pigments, protein contents and HMGR enzyme activity was also noticed in transgenic plants over control. Interestingly, the latex yield was significantly enhanced in all transgenic plants compared to the control. The qRT-PCR results exhibit that the hmgr1 mRNA transcript levels was 160-fold more abundance in transgenic plants over untransformed control. These results altogether suggest that there is a positive correlation between latex yield and accumulation of mRNA transcripts level as well as HMGR enzyme activity in transgenic rubber plants. It is presumed that there is a possibility for enhanced level of latex biosynthesis in transgenic plants as the level of mRNA transcripts and HMGR enzyme activity is directly correlated with latex yield in rubber tree. Further, the present results clearly suggest that the quantification of HMGR enzyme activity in young seedlings will be highly beneficial for early selection of high latex yielding plants in rubber breeding programs. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Identification of viral and non-viral reverse transcribing elements in pineapple (Ananas comosus), including members of two new badnavirus species.

PubMed

Gambley, C F; Geering, A D W; Steele, V; Thomas, J E

2008-01-01

A previously published partial sequence of pineapple bacilliform virus was shown to be from a retrotransposon (family Metaviridae) and not from a badnavirus as previously thought. Two newly discovered sequence groups isolated from pineapple were associated with bacilliform virions and were transmitted by mealybugs. Phylogenetic analyses indicated that they were members of new badnavirus species. A third caulimovirid sequence was also amplified from pineapple, but available evidence suggests that this DNA is not encapsidated, but more likely derived from an endogenous virus.

New Insights for Diagnosis of Pineapple Fusariosis by MALDI-TOF MS Technique.

PubMed

Santos, Cledir; Ventura, José Aires; Lima, Nelson

2016-08-01

Fusarium is one of the most economically important fungal genus, since it includes many pathogenic species which cause a wide range of plant diseases. Morphological or molecular biology identification of Fusarium species is a limiting step in the fast diagnosis and treatment of plant disease caused by these fungi. Mass spectrometry by matrix-assisted laser/desorption ionisation-time-of-flight (MALDI-TOF)-based fingerprinting approach was applied to the fungal growth monitoring and direct detection of strain Fusarium guttiforme E-480 inoculated in both pineapple cultivars Pérola and Imperial side shoots, that are susceptible and resistant, respectively, to this fungal strain. MALDI-TOF MS technique was capable to detect fungal molecular mass peaks in the susceptible pineapple stem side shoot tissue. It is assumed that these molecular masses are mainly constituted by ribosomal proteins. MALDI-TOF-based fingerprinting approach has herein been demonstrated to be sensitive and accurate for the direct detection of F. guttiforme E-480 molecular masses on both susceptible and resistant pineapple side stem free of any pre-treatment. According to the results obtained, the changing on molecular mass peaks of infected susceptible pineapple tissue together with the possibility of fungal molecular masses analysis into this pineapple tissue can be a good indication for an early diagnosis by MALDI-TOF MS of pineapple fusariosis.

Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange (Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker disease.

PubMed

Sendín, Lorena Noelia; Orce, Ingrid Georgina; Gómez, Rocío Liliana; Enrique, Ramón; Grellet Bournonville, Carlos Froilán; Noguera, Aldo Sergio; Vojnov, Adrián Alberto; Marano, María Rosa; Castagnaro, Atilio Pedro; Filippone, María Paula

2017-04-01

Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

Production of hydroxylated fatty acids in genetically modified plants

DOEpatents

Somerville, Chris [Portola Valley, CA; Broun, Pierre [Burlingame, CA; van de Loo, Frank [Weston, AU; Boddupalli, Sekhar S [Manchester, MI

2011-08-23

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

Production of hydroxylated fatty acids in genetically modified plants

DOEpatents

Somerville, Chris; Broun, Pierre; van de Loo, Frank; Boddupalli, Sekhar S.

2005-08-30

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

Validation of a recombinant human bactericidal/permeability-increasing protein (hBPI) expression vector using murine mammary gland tumor cells and the early development of hBPI transgenic goat embryos.

PubMed

Gui, Tao; Liu, Xing; Tao, Jia; Chen, Jianwen; Li, Yunsheng; Zhang, Meiling; Wu, Ronghua; Zhang, Yuanliang; Peng, Kaisong; Liu, Ya; Zhang, Xiaorong; Zhang, Yunhai

2013-12-01

Human bactericidal/permeability-increasing protein (hBPI) is the only antibacterial peptide which acts against both gram-negative bacteria and neutralizes endotoxins in human polymorphonuclear neutrophils; therefore, hBPI is of great value in clinical applications. In the study, we constructed a hBPI expression vector (pBC1-Loxp-Neo-Loxp-hBPI) containing the full-length hBPI coding sequence which could be specifically expressed in the mammary gland. To validate the function of the vector, in vitro cultured C127 (mouse mammary Carcinoma Cells) were transfected with the vector, and the transgenic cell clones were selected to express hBPI by hormone induction. The mRNA and protein expression of hBPI showed that the constructed vector was effective and suitable for future application in producing mammary gland bioreactor. Then, female and male goat fibroblasts were transfected with the vector, and two male and two female transgenic clonal cell lines were obtained. Using the transgenic cell lines as nuclear donors for somatic cell nuclear transfer, the reconstructed goat embryos produced from all four clones could develop to blastocysts in vitro. In conclusion, we constructed and validated an efficient mammary gland-specific hBPI expression vector, pBC1-Loxp-Neo-Loxp-hBPI, and transgenic hBPI goat embryos were successfully produced, laying foundations for future production of recombinant hBPI in goat mammary gland. Copyright © 2013 Elsevier B.V. All rights reserved.

[Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

PubMed

Li, Lan; Shen, Wei; Pan, Qing-Yu; Min, Ling-Jiang; Sun, Yu-Jiang; Fang, Yong-Wei; Deng, Ji-Xian; Pan, Qing-Jie

2006-12-01

Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

Metabolite profiling and volatiles of pineapple wine and vinegar obtained from pineapple waste.

PubMed

Roda, Arianna; Lucini, Luigi; Torchio, Fabrizio; Dordoni, Roberta; De Faveri, Dante Marco; Lambri, Milena

2017-08-15

Vinegar is an inexpensive commodity, and economic considerations require that a relatively low-cost raw material be used for its production. An investigation into the use of a new, alternative substrate - pineapple waste - is described. This approach enables the utilization of the pineapple's (Ananas comosus) peels and core, which are usually discarded during the processing or consumption of the fruit. Using physical and enzymatic treatments, the waste was saccharified, and the resulting substrate was fermented with Saccharomyces cerevisiae for 7-10days under aerobic conditions at 25°C. This resulted in an alcohol yield of approximately 7%. The alcoholic medium was then used as a seed broth for acetic fermentation using Acetobacter aceti as the inoculum for approximately 30days at 32°C to obtain 5% acetic acid. Samples were analyzed at the beginning and end of the acetification cycle to assess the volatile and fixed compounds by GC-MS and UHPLC-QTOF-MS. The metabolomic analysis indicated that l-lysine, mellein, and gallic acid were significantly more concentrated in the pineapple vinegar than in the original wine. Higher alcohols, aldehydes, and ketones characterized the aroma of the final pineapple vinegar, whilst off-flavors were significantly reduced relative to the initial wine. This study is the first to highlight the metabolite profile of fruit vinegar with a slight floral aroma profile derived from pineapple waste. The potential to efficiently reduce the post-harvest losses of pineapple fruits by re-using them for products with added food values is also demonstrated. Copyright © 2017 Elsevier Ltd. All rights reserved.

A modular toolset for recombination transgenesis and neurogenetic analysis of Drosophila.

PubMed

Wang, Ji-Wu; Beck, Erin S; McCabe, Brian D

2012-01-01

Transgenic Drosophila have contributed extensively to our understanding of nervous system development, physiology and behavior in addition to being valuable models of human neurological disease. Here, we have generated a novel series of modular transgenic vectors designed to optimize and accelerate the production and analysis of transgenes in Drosophila. We constructed a novel vector backbone, pBID, that allows both phiC31 targeted transgene integration and incorporates insulator sequences to ensure specific and uniform transgene expression. Upon this framework, we have built a series of constructs that are either backwards compatible with existing restriction enzyme based vectors or utilize Gateway recombination technology for high-throughput cloning. These vectors allow for endogenous promoter or Gal4 targeted expression of transgenic proteins with or without fluorescent protein or epitope tags. In addition, we have generated constructs that facilitate transgenic splice isoform specific RNA inhibition of gene expression. We demonstrate the utility of these constructs to analyze proteins involved in nervous system development, physiology and neurodegenerative disease. We expect that these reagents will facilitate the proficiency and sophistication of Drosophila genetic analysis in both the nervous system and other tissues.

First report of Pineapple mealybug wilt associated virus-1 in Ecuador

USDA-ARS?s Scientific Manuscript database

In Ecuador, where pineapple represents one of the most important export commodities, virus testing has been neglected. In July 2014, a total of twenty MD2 hybrid pineapple plants showing virus-like symptoms (Fig. 1) were collected from a commercial planting located at the border of Santo Domingo and...

Effects of pineapple byproduct and canola oil as fat replacers on physicochemical and sensory qualities of low-fat beef burger.

PubMed

Selani, Miriam M; Shirado, Giovanna A N; Margiotta, Gregório B; Saldaña, Erick; Spada, Fernanda P; Piedade, Sonia M S; Contreras-Castillo, Carmen J; Canniatti-Brazaca, Solange G

2016-02-01

Pineapple byproduct and canola oil were evaluated as fat replacers on physicochemical and sensory characteristics of low-fat burgers. Five treatments were performed: conventional (CN, 20% fat) and four low-fat formulations (10% fat): control (CT), pineapple byproduct (PA), canola oil (CO), pineapple byproduct and canola oil (PC). Higher water and fat retention and lower cooking loss and diameter reduction were found in burgers with byproduct addition. In raw burgers, byproduct incorporation reduced L*, a*, and C* values, but these alterations were masked after cooking, leading to products similar to CN. Low-fat treatments were harder, chewier, and more cohesive than full-fat burgers. However, in Warner Bratzler shear measurements, PA and PC were as tender as CN. In QDA, no difference was found between CN and PC. Pineapple byproducts along with canola oil are promising fat replacers in beef burgers. In order to increase the feasibility of use of pineapple byproduct in the meat industry, alternative processes of byproduct preparation should be evaluated in future studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genome-wide identification, functional and evolutionary analysis of terpene synthases in pineapple.

PubMed

Chen, Xiaoe; Yang, Wei; Zhang, Liqin; Wu, Xianmiao; Cheng, Tian; Li, Guanglin

2017-10-01

Terpene synthases (TPSs) are vital for the biosynthesis of active terpenoids, which have important physiological, ecological and medicinal value. Although terpenoids have been reported in pineapple (Ananas comosus), genome-wide investigations of the TPS genes responsible for pineapple terpenoid synthesis are still lacking. By integrating pineapple genome and proteome data, twenty-one putative terpene synthase genes were found in pineapple and divided into five subfamilies. Tandem duplication is the cause of TPS gene family duplication. Furthermore, functional differentiation between each TPS subfamily may have occurred for several reasons. Sixty-two key amino acid sites were identified as being type-II functionally divergence between TPS-a and TPS-c subfamily. Finally, coevolution analysis indicated that multiple amino acid residues are involved in coevolutionary processes. In addition, the enzyme activity of two TPSs were tested. This genome-wide identification, functional and evolutionary analysis of pineapple TPS genes provide a new insight into understanding the roles of TPS family and lay the basis for further characterizing the function and evolution of TPS gene family. Copyright © 2017 Elsevier Ltd. All rights reserved.

The physiology of ex vitro pineapple (Ananas comosus L. Merr. var MD-2) as CAM or C3 is regulated by the environmental conditions: proteomic and transcriptomic profiles.

PubMed

Aragón, C; Pascual, P; González, J; Escalona, M; Carvalho, L; Amancio, S

2013-11-01

Proteomic and transcriptomic profiles of key enzymes were monitored in pineapple plants propagated under C3 and CAM-inducing metabolisms to obtain insight into the CAM-facultative metabolism and the relationship of CAM plants with oxidative stress. Pineapple is one of the most important tropical crops worldwide. The use of temporary immersion bioreactors for the first stages of pineapple propagation enables precise control of plant growth, increases the rate of plant multiplication, decreases space, energy and labor requirements for pineapple plants in commercial micropropagation. Once the plantlets are ready to be taken from the reactors, they are carefully acclimatized to natural environmental conditions, and a facultative C3/CAM metabolism in the first 2 months of growth is the characteristic of pineapple plants, depending on environmental conditions. We subjected two sets of micropropagated pineapple plants to C3 and CAM-inducing environmental conditions, determined by light intensity/relative humidity (respectively 40 μmol m−2 s−1/85 % and 260 μmol m−2 s−1/50 %). Leaves of pineapple plants grown under CAM-inducing conditions showed higher leaf thickness and more developed cuticles and hypodermic tissue. Proteomic profiles of several proteins, isoenzyme patterns and transcriptomic profiles were also measured. Five major spots were isolated and identified, two of them for the first time in Ananas comosus (OEE 1; OEE 2) and the other three corresponding to small fragments of the large subunit of Rubisco (LSU). PEPC and PEPCK were also detected by immunobloting of 2DE at the end of both ex vitro treatments (C3/CAM) during the dark period. Isoenzymes of SOD and CAT were identified by electrophoresis and the transcript levels of OEE 1 and CAT were associated with CAM metabolism in pineapple plants.

Dietary supplementation with fresh pineapple juice decreases inflammation and colonic neoplasia in IL-10-deficient mice with colitis.

PubMed

Hale, Laura P; Chichlowski, Maciej; Trinh, Chau T; Greer, Paula K

2010-12-01

Bromelain, a mixture of proteolytic enzymes typically derived from pineapple stem, decreases production of proinflammatory cytokines and leukocyte homing to sites of inflammation. We previously showed that short-term oral treatment with bromelain purified from pineapple stem decreased the severity of colonic inflammation in C57BL/6 Il10(-/-) mice with chronic colitis. Since fresh pineapple fruit contains similar bromelain enzymes but at different proportions, this study aimed to determine whether long-term dietary supplementation with pineapple (supplied as juice) could decrease colon inflammation and neoplasia in Il10(-/-) mice with chronic colitis as compared with bromelain derived from stem. Colitis was triggered in Il10(-/-) mice by exposure to the non-steroidal anti-inflammatory drug piroxicam. Mice with colitis were supplemented with fresh vs. boiled pineapple juice or bromelain purified from stem for up to 6 months. Experimental mice readily consumed fresh pineapple juice at a level that generated mean stool proteolytic activities equivalent to 14 mg bromelain purified from stem, while control mice received boiled juice with inactive enzymes. Survival was increased in the group supplemented with fresh rather than boiled juice (P = 0.01). Mice that received fresh juice also had decreased histologic colon inflammation scores and a lower incidence of inflammation-associated colonic neoplasia (35% versus 66%; P < 0.02), with fewer neoplastic lesions/colon (P = 0.05). Flow cytometric analysis of murine splenocytes exposed to fresh pineapple juice in vitro demonstrated proteolytic removal of cell surface molecules that can affect leukocyte trafficking and activation. These results demonstrate that long-term dietary supplementation with fresh or unpasteurized frozen pineapple juice with proteolytically active bromelain enzymes is safe and decreases inflammation severity and the incidence and multiplicity of inflammation-associated colonic neoplasia in this commonly used murine model of inflammatory bowel disease. Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.

Biohydrogen Production from Pineapple Waste: Effect of Substrate Concentration and Acid Pretreatment

NASA Astrophysics Data System (ADS)

Cahyari, K.; Putri, A. M.; Oktaviani, E. D.; Hidayat, M. A.; Norajsha, J. D.

2018-05-01

Biohydrogen is the ultimate choice of energy carrier in future due to its superior qualities such as fewer greenhouse gases emission, high energy density (142 kJ/gram), and high energy conversion using a fuel cell. Production of biohydrogen from organic waste e.g. pineapple waste offers a simultaneous solution for renewable energy production and waste management. It is estimated that pineapple cultivation in Indonesia generated more than 1 million ton/year comprising of rotten pineapple fruit, leaves, and stems. Majority of this waste is dumped into landfill area without any treatments which lead to many environmental problems. This research was meant to investigate the utilization of pineapple waste i.e. peel and the core of pineapple fruit and leaves to produce biohydrogen through mesophilic dark fermentation (30°C, 1 atm, pH 5.0). Effect of dilute acid treatment and substrate concentration was particularly investigated in these experiments. Peel and core of pineapple waste were subjected to fermentation at 3 various substrate concentration i.e. 8.8, 17.6 and 26.4-gram VS/liter. Meanwhile, pineapple leaves were pretreated using dilute acid (H2SO4) at 0.2, 0.3 and 0.4 N and followed by dark fermentation. Results show that the highest yield of biohydrogen was obtained at a substrate concentration of 26.4-gram VS/liter both for peel and core of the waste. Pretreatment using dilute acid (H2SO4) 0.3 N might improve fermentation process with a higher yield at 0.8 ml/gram VS. Hydrogen percentage in biogas produced during fermentation process was in the range between 5 – 32% of volume ratio. In summary, it is possible to utilize pineapple waste for production of biohydrogen at an optimum substrate concentration of 26.4-gram VS/liter and acid pretreatment (H2SO4) of 0.3 N.

The database on transgenic luminescent microorganisms as an instrument of studying a microbial component of closed ecosystems

NASA Astrophysics Data System (ADS)

Boyandin, A. N.; Lankin, Y. P.; Kargatova, T. V.; Popova, L. Y.; Pechurkin, N. S.

Luminescent transgenic microorganisms are widely used for study of microbial communities' functioning including closed ones. Bioluminescence is of high sensitive to effects of different environmental factors. Integration of lux-genes into different metabolic ways allows studying many aspects of microorganisms' life permitting to carry out measurements in situ. There is much information about applications of bioluminescent bacteria in different researches. But for effective using these data their summarizing and accumulation in common source is required. Therefore an information system on characteristics of transgenic microorganisms with cloned lux-genes was created. The database and client software related were developed. A database structure includes information on common characteristics of cloned lux-genes, their sources and properties, on regulation of gene expression in bacterial cells, on dependence of bioluminescence manifestation on biotic, abiotic and anthropogenic environmental factors. The database also can store description of changes in bacterial populations depending on environmental changes. The database created allows storing and using bibliographic information and also links to web sites of world collections of microorganisms. Internet publishing software permitting to open access to the database through the Internet is developed.

Expression of an alfalfa (Medicago sativa L.) peroxidase gene in transgenic Arabidopsis thaliana enhances resistance to NaCl and H2O2.

PubMed

Teng, K; Xiao, G Z; Guo, W E; Yuan, J B; Li, J; Chao, Y H; Han, L B

2016-05-23

Peroxidases (PODs) are enzymes that play important roles in catalyzing the reduction of H2O2 and the oxidation of various substrates. They function in many different and important biological processes, such as defense mechanisms, immune responses, and pathogeny. The POD genes have been cloned and identified in many plants, but their function in alfalfa (Medicago sativa L.) is not known, to date. Based on the POD gene sequence (GenBank accession No. L36157.1), we cloned the POD gene in alfalfa, which was named MsPOD. MsPOD expression increased with increasing H2O2. The gene was expressed in all of the tissues, including the roots, stems, leaves, and flowers, particularly in stems and leaves under light/dark conditions. A subcellular analysis showed that MsPOD was localized outside the cells. Transgenic Arabidopsis with MsPOD exhibited increased resistance to H2O2 and NaCl. Moreover, POD activity in the transgenic plants was significantly higher than that in wild-type Arabidopsis. These results show that MsPOD plays an important role in resistance to H2O2 and NaCl.

Acceptability of minimally processed and irradiated pineapple and watermelon among Brazilian consumers

NASA Astrophysics Data System (ADS)

Martins, Cecília Geraldes; Aragon-Alegro, Lina Casale; Behrens, Jorge Herman; Oliveira Souza, Kátia Leani; Martins Vizeu, Dirceu; Hutzler, Beatriz Weltman; Teresa Destro, Maria; Landgraf, Mariza

2008-06-01

This study aimed at evaluating the acceptance of MP watermelon and pineapple exposed to 1.0 and 2.5 kGy compared to non-irradiated samples. No significant differences were observed in liking between irradiated and non-irradiated samples, and also between doses of 1.0 and 2.5 kGy. Significant differences in sourness (pineapple) or sweetness (watermelon) and between intention of purchase of irradiated and non-irradiated fruits were not observed as well. Results showed that MP watermelon and pineapple could be irradiated with doses up to 2.5 kGy without significant changes in acceptability.

Goat RSPO1 over-expression rescues sex-reversal in Rspo1-knockout XX mice but does not perturb testis differentiation in XY or sex-reversed XX mice.

PubMed

Buscara, Laurine; Montazer-Torbati, Fatemeh; Chadi, Sead; Auguste, Aurélie; Laubier, Johann; Chassot, Anne-Amandine; Renault, Lauriane; Passet, Bruno; Costa, José; Pannetier, Maëlle; Vilotte, Marthe; Chaboissier, Marie-Christine; Vilotte, Jean-Luc; Pailhoux, Eric; Le Provost, Fabienne

2009-08-01

RSPO1 is a newly discovered gene involved in sex differentiation. Two goat BAC clones encompassing the RSPO1 gene (gRSPO1) were injected into mouse oocytes and several transgenic lines derived. Both clones induced gRSPO1 over-expression in various tissues, including male and female gonads, with no obvious phenotype and normal sex-ratios. Introgression of the gRSPO1 transgene into a mouse RSPO1 knockout genotype resulted in the rescue of the fertility and the disappearance of the masculinized gonadic features of the females, demonstrating the functionality of the goat protein in a mouse context. On the contrary, over-expression of gRSPO1 within a mSRY or a gSRY-XX genotypes did not interfere with the SRY-induced male phenotype.

Production of Multiple Transgenic Yucatan Miniature Pigs Expressing Human Complement Regulatory Factors, Human CD55, CD59, and H-Transferase Genes

PubMed Central

Jang, Gun-Hyuk; Jeong, Yeun-Ik; Hwang, In-Sung; Jeong, Yeon-woo; Kim, Yu-Kyung; Shin, Taeyoung; Kim, Nam-Hyung; Hyun, Sang-Hwan; Jeung, Eui-Bae; Hwang, Woo-Suk

2013-01-01

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications. PMID:23704897

Production of transgenic cattle highly expressing human serum albumin in milk by phiC31 integrase-mediated gene delivery.

PubMed

Luo, Yan; Wang, Yongsheng; Liu, Jun; Lan, Hui; Shao, Minghao; Yu, Yuan; Quan, Fusheng; Zhang, Yong

2015-10-01

Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs). PhiC31 integrase-mediated integrations in genome of BFFs were screened by nested inverse PCR. After analysis of sequence of the PCR products, 46.0% (23/50) of the both attB-genome junction sites (attL and attR) were confirmed, and four pseudo attP sites were identified. The integration rates in BF3, BF11, BF19 and BF4 sites were 4.0% (2/50), 6.0% (3/50), 16.0% (8/50) and 20.0% (10/50), respectively. BF3 is located in the bovine chromosome 3 collagen alpha-3 (VI) chain isomer 2 gene, while the other three sites are located in the non-coding region. The transgenic cell lines from BF11, BF19 and BF4 sites were used as donors for SCNT. Two calves from transgenic cells BF19 were born, one died within a few hours after birth, and another calf survived healthy. PCR and Southern blot analysis revealed integration of the transgene in the genome of cloned calves. The nested reverse PCR confirmed that the integration site in cloned calves was identical to the donor cells. The western blotting assessment indicated that recombinant HSA was expressed in the milk of transgenic cattle and the expression level was about 4-8 mg/mL. The present study demonstrated that phiC31 integrase system was an efficient and safety gene delivery tool for producing HSA transgenic cattle. The production of recombinant HSA in the milk of cattle may provide a large-scale and cost-effective resource.

Genomic analyses of the CAM plant pineapple.

PubMed

Zhang, Jisen; Liu, Juan; Ming, Ray

2014-07-01

The innovation of crassulacean acid metabolism (CAM) photosynthesis in arid and/or low CO2 conditions is a remarkable case of adaptation in flowering plants. As the most important crop that utilizes CAM photosynthesis, the genetic and genomic resources of pineapple have been developed over many years. Genetic diversity studies using various types of DNA markers led to the reclassification of the two genera Ananas and Pseudananas and nine species into one genus Ananas and two species, A. comosus and A. macrodontes with five botanical varieties in A. comosus. Five genetic maps have been constructed using F1 or F2 populations, and high-density genetic maps generated by genotype sequencing are essential resources for sequencing and assembling the pineapple genome and for marker-assisted selection. There are abundant expression sequence tag resources but limited genomic sequences in pineapple. Genes involved in the CAM pathway has been analysed in several CAM plants but only a few of them are from pineapple. A reference genome of pineapple is being generated and will accelerate genetic and genomic research in this major CAM crop. This reference genome of pineapple provides the foundation for studying the origin and regulatory mechanism of CAM photosynthesis, and the opportunity to evaluate the classification of Ananas species and botanical cultivars. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Effect of UV-C radiation on bioactive compounds of pineapple (Ananas comosus L. Merr.) by-products.

PubMed

Freitas, Ana; Moldão-Martins, Margarida; Costa, Helena S; Albuquerque, Tânia G; Valente, Ana; Sanches-Silva, Ana

2015-01-01

The industrial processing of pineapple generates a high quantity of by-products. To reduce the environmental impact of these by-products and the inherent cost of their treatment, it is important to characterise and valorise these products, converting them into high added value products. Ultra-violet radiation is one of the main sustainable sanitation techniques for fruits. Since this radiation can induce plant stress which can promote the biosynthesis of bioactive compounds, it is important to evaluate its effect in fruits. The amounts of vitamins (C and E) and carotenoids (α-carotene, β-carotene, β-cryptoxanthin, lutein, lycopene, neoxanthin, violaxanthin and zeaxanthin) in pineapple by-products (core and rind) were analysed before and after treatment with UV radiation. All treated and untreated pineapple by-products contained β-carotene as the main carotenoid (rind, 2537-3225 µg; and core, 960-994 µg 100 g(-1) DW). Pineapple rind also contained lutein (288-297 µg 100 g(-1) DW) and α-carotene (89-126 µg 100 g(-1) DW). The results provide evidence of the potential of pineapple by-products as a source of bioactive compounds with antioxidant activity, which can be used by pharmaceutical, cosmetics and food industries. In addition, UV-C was shown to be a treatment that can add nutritional value to pineapple by-products. © 2014 Society of Chemical Industry.

Ethical issues in transgenics.

PubMed

Sherlock, R; Morrey, J D

2000-01-01

The arguments of critics and concerns of the public on generating transgenic cloned animals are analyzed for the absence or presence of logical structure. Critics' arguments are symbolically compared with "genetic trespassing," "genetic speeding," or "going the wrong way," and responses are provided to these arguments. Scientists will be empowered to participate in the public discussion and to engage the critics on these issues as they consider thoughtful, plausible responses to their concerns. Temporary moratoriums are recognized as a plausible approach to dealing with possible concerns of new scientific advancements.

Evaluating the Genetic, Hormonal, and Exogenous Factors Affecting Somatic Copy Number Variation in Breast Cancer

DTIC Science & Technology

2016-10-01

progress in subaim 1a, substantially improving the design of our proposed transgenic animal, the “deletion reporter mouse”, and are finalizing cloning...of necessary components. We expect to submit embryonic stem cells to the transgenic facility within the next few months. Furthermore, subaim 1b is...different mammary epithelial subpopulations. We will breed the reporter mouse created in aim 1 (or the CAG/UBC-GFP mouse) with BRCA1+/- and ATM+/- mutant

Biology and trapping of stable flies (Diptera: Muscidae) developing in pineapple residues (Ananas comosus) in Costa Rica

USDA-ARS?s Scientific Manuscript database

Pineapple production in Costa Rica increased nearly 300-fold during the last 30 yr, and >40,000 hectares of land are currently dedicated to this crop. At the end of the pineapple cropping cycle, plants are chopped and residues incorporated into the soil in preparation for replanting. Associated with...

Positive Selection of γδ CTL by TL Antigen Expressed in the Thymus

PubMed Central

Tsujimura, Kunio; Takahashi, Toshitada; Morita, Akimichi; Hasegawa-Nishiwaki, Hitomi; Iwase, Shigeru; Obata, Yuichi

1996-01-01

To elucidate the function of the mouse TL antigen in the thymus, we have derived two TL transgenic mouse strains by introducing Tla a -3 of A strain origin with its own promoter onto a C3H background with no expression of TL in the thymus. These transgenic mouse strains, both of which express high levels of Tlaa-3-TL antigen in their thymus, were analyzed for their T cell function with emphasis on cytotoxic T lymphocyte (CTL) generation. A T cell response against TL was induced in Tg.Tlaa-3-1, Tg.Tlaa-3-2, and control C3H mice by skin grafts from H-2K b/T3 b transgenic mice, Tg.Con.3-1, expressing T3b-TL ubiquitously. Spleen cells from mice that had rejected the T3b-TL positive skin grafts were restimulated in vitro with Tg.Con.3-1 irradiated spleen cells. In mixed lymphocyte cultures (MLC), approximately 20% and 15% of Thy-1+ T cells derived from Tg.Tlaa-3-1 and Tg.Tlaa-3-2, respectively, expressed TCRγδ, whereas almost all those from C3H expressed TCRαβ. The MLC from Tg.Tlaa-3-2 and C3H demonstrated high CTL activity against TL, while those from Tg.Tlaa-3-1 had little or none. The generation of γδ CTL recognizing TL in Tg.Tlaa-3-2, but not C3H mice, was confirmed by the establishment of CTL clones. A total of 14 γδ CTL clones were established from Tg.Tlaa-3-2, whereas none were obtained from C3H. Of the 14 γδ CTL clones, 8 were CD8+ and 6 were CD4−CD8− double negative. The CTL activity of all these clones was TL specific and inhibited by anti-TL, but not by anti-H-2 antibodies, demonstrating that they recognize TL directly without antigen presentation by H-2. The CTL activity was blocked by antibodies to TCRγδ and CD3, and also by antibodies to CD8α and CD8β in CD8+ clones, showing that the activity was mediated by TCRγδ and coreceptors. The thymic origin of these γδ CTL clones was indicated by the expression of Thy-1 and Ly-1 (CD5), and also CD8αβ heterodimers in CD8+ clones on their surfaces and by the usage of TCR Vγ4 chains in 12 of the 14 clones. Taken together, these results suggest that Tlaa-3-TL antigen expressed in the thymus engages in positive selection of a sizable population of γδ T cells. PMID:8976173

Bioprospecting of powdered pineapple rind as an organic supplement of composted sawdust for Pleurotus ostreatus mushroom cultivation.

PubMed

Narh Mensah, Deborah L; Addo, Peter; Dzomeku, Matilda; Obodai, Mary

2018-03-01

Pineapple rind is a by-product of the pineapple processing industry and contains nutrients and other compounds which must be utilized as a bioresource for socio-economic benefits while preventing the potential problems of improper agroindustrial biomass disposal methods. Pleurotus ostreatus is an edible oyster mushroom with medicinal properties and can be cultivated on various agroindustrial biomass, including sawdust containing supplements. Pineapple rind was powdered and used as a supplement of composted sawdust at 2%, 5%, 10%, 12%, 15%, and 20% (w/w) on dry weight basis. A control treatment consisted of composted sawdust supplemented with rice bran at 12% (the most utilized composition in Ghana). P. ostreatus strain EM-1 was cultivated on these treatments. Factors investigated included the spawn run period, yield, fruiting body weight and size, biological efficiency, and nutritional composition (proximate composition and Copper, Zinc and Lead content) of fruiting bodies harvested from selected high-yielding treatments and the control treatment. Full colonization of all treatments occurred by the 34th day of incubation. Enhanced yield, fruiting body weight and size, and biological efficiency were generally recorded with supplementation at lower concentrations (2% and 5%) compared to treatments supplemented at higher concentrations. There was also a supplement concentration-dependent alteration of the nutritional composition of the mushroom. Powdered pineapple rind can be utilized as an organic supplement at relatively low concentrations in composted sawdust for P. ostreatus strain EM-1 cultivation. The use of lower concentrations of powdered pineapple rind in composted sawdust is advantageous as relatively less input will be required to produce higher P. ostreatus strain EM-1 yields. Utilization of pineapple rind for mushroom cultivation will extend the pineapple plant value chain, intensify mushroom production in a sustainable way, and minimize agricultural losses.

Specificity of monoclonal antibodies to strains of Dickeya sp. that cause bacterial heart rot of pineapple.

PubMed

Peckham, Gabriel D; Kaneshiro, Wendy S; Luu, Van; Berestecky, John M; Alvarez, Anne M

2010-10-01

During a severe outbreak of bacterial heart rot that occurred in pineapple plantations on Oahu, Hawaii, in 2003 and years following, 43 bacterial strains were isolated from diseased plants or irrigation water and identified as Erwinia chrysanthemi (now Dickeya sp.) by phenotypic, molecular, and pathogenicity assays. Rep-PCR fingerprint patterns grouped strains from pineapple plants and irrigation water into five genotypes (A-E) that differed from representatives of other Dickeya species, Pectobacterium carotovorum and other enteric saprophytes isolated from pineapple. Monoclonal antibodies produced following immunization of mice with virulent type C Dickeya sp. showed only two specificities. MAb Pine-1 (2D11G1, IgG1 with kappa light chain) reacted to all 43 pineapple/water strains and some reference strains (D. dianthicola, D. chrysanthemi, D. paradisiaca, some D. dadantii, and uncharacterized Dickeya sp.) but did not react to reference strains of D. dieffenbachiae, D. zeae, or one of the two Malaysian pineapple strains. MAb Pine-2 (2A7F2, IgG3 with kappa light chain) reacted to all type B, C, and D strains but not to any A or E strains or any reference strains except Dickeya sp. isolated from Malaysian pineapple. Pathogenicity tests showed that type C strains were more aggressive than type A strains when inoculated during cool months. Therefore, MAb Pine-2 distinguishes the more virulent type C strains from less virulent type A pineapple strains and type E water strains. MAbs with these two specificities enable development of rapid diagnostic tests that will distinguish the systemic heart rot pathogen from opportunistic bacteria associated with rotted tissues. Use of the two MAbs in field assays also permits the monitoring of a known subpopulation and provides additional decision tools for disease containment and management practices.

Respiratory properties and malate metabolism in Percoll-purified mitochondria isolated from pineapple, Ananas comosus (L.) Merr. cv. smooth cayenne.

PubMed

Hong, Hoang Thi Kim; Nose, Akihiro; Agarie, Sakae

2004-10-01

An investigation was made of the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant Ananas comosus (pineapple) in malate metabolism during CAM phase III. Pineapple mitochondria showed very high malate dehydrogenase (MDH), and low malic enzyme (ME) and glutamate-oxaloacetate transaminase (GOT) activities. The mitochondria readily oxidized succinate and NADH with high rates and coupling, while they only oxidized NADPH in the presence of Ca(2+). Pineapple mitochondria oxidized malate with low rates under most assay conditions, despite increasing malate concentrations, optimizing pH, providing cofactors such as coenzyme A, thiamine pyrophosphate, and NAD(+), and supplying individually external glutamate or GOT. However, providing glutamate and GOT simultaneously strongly increased the rates of malate oxidation. The OAA easily permeated the mitochondrial membranes to import into or export out of pineapple mitochondria during malate oxidation, but the mitochondria did not consume external Asp or alpha-KG. These results suggest that OAA played a significant role in the mitochondrial malate metabolism of pineapple, in which malate was mainly oxidized by active mMDH to produce OAA which could be exported outside the mitochondria via a malate-OAA shuttle. Cytosolic GOT then consumed OAA by transamination in the presence of glutamate, leading to a large increase in respiration rates. The malate-OAA shuttle might operate as a supporting system for decarboxylation in phase III of PCK-CAM pineapple. This shuttle system may be important in pineapple to provide a source of energy and substrate OAA for cytosolic PCK activity during the day when cytosolic OAA and ATP was limited for the overall decarboxylation process.

The physiology of ex vitro pineapple (Ananas comosus L. Merr. var MD-2) as CAM or C3 is regulated by the environmental conditions.

PubMed

Aragón, C; Carvalho, L; González, J; Escalona, M; Amancio, S

2012-04-01

Many plant species grown under in vitro controlled conditions can be used as models for the study of physiological processes. Adult pineapple can display CAM physiology while in vitro it functions as a C3 plant. Ex vitro Ananas comosus has plastic morphology and physiology, both easy to modify from C3 to CAM by changing the environmental conditions. The yield of survival for a rentable propagation protocol of pineapple is closely related with the C3/CAM shift and the associated physiological characteristics. In the present work, ex vitro pineapple plants were divided in two sets and subjected to C3 and CAM-inducing environmental conditions, determined by light intensity and relative humidity, respectively, 40 μmol m(-2) s(-1)/85% and 260 μmol m(-2) s(-1)/50%. The results demonstrated that the stress imposed by the environmental conditions switched pineapple plants from C3 to CAM behavior. Comparing to CAM induced, C3-induced pineapple plants showed substandard growth parameters and morphological leaf characteristics but a better rooting process and a higher ABA production, a phenotype closer to adult plants, which are expected to produce fruits in a normal production cycle. We conclude that the upholding of these characteristics is conditioned by low light intensity plus high relative humidity, especially during the first 8 weeks of ex vitro growth. It is expected that the better understanding of pineapple acclimatization will contribute to the design of a protocol to apply as a rentable tool in the pineapple agronomic industry. © Springer-Verlag 2011

The pineapple genome and the evolution of CAM photosynthesis

PubMed Central

Ming, Ray; VanBuren, Robert; Wai, Ching Man; Tang, Haibao; Schatz, Michael C.; Bowers, John E.; Lyons, Eric; Wang, Ming-Li; Chen, Jung; Biggers, Eric; Zhang, Jisen; Huang, Lixian; Zhang, Lingmao; Miao, Wenjing; Zhang, Jian; Ye, Zhangyao; Miao, Chenyong; Lin, Zhicong; Wang, Hao; Zhou, Hongye; Yim, Won C.; Priest, Henry D.; Zheng, Chunfang; Woodhouse, Margaret; Edger, Patrick P.; Guyot, Romain; Guo, Hao-Bo; Guo, Hong; Zheng, Guangyong; Singh, Ratnesh; Sharma, Anupma; Min, Xiangjia; Zheng, Yun; Lee, Hayan; Gurtowski, James; Sedlazeck, Fritz J.; Harkess, Alex; McKain, Michael R.; Liao, Zhenyang; Fang, Jingping; Liu, Juan; Zhang, Xiaodan; Zhang, Qing; Hu, Weichang; Qin, Yuan; Wang, Kai; Chen, Li-Yu; Shirley, Neil; Lin, Yann-Rong; Liu, Li-Yu; Hernandez, Alvaro G.; Wright, Chris L.; Bulone, Vincent; Tuskan, Gerald A.; Heath, Katy; Zee, Francis; Moore, Paul H.; Sunkar, Ramanjulu; Leebens-Mack, James H.; Mockler, Todd; Bennetzen, Jeffrey L.; Freeling, Michael; Sankoff, David; Paterson, Andrew H.; Zhu, Xinguang; Yang, Xiaohan; Smith, J. Andrew C.; Cushman, John C.; Paull, Robert E.; Yu, Qingyi

2016-01-01

Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water use efficiency, and the second most important tropical fruit after banana in terms of international trade. We sequenced the genomes of pineapple varieties ‘F153’ and ‘MD2’, and a wild pineapple relative A. bracteatus accession CB5. The pineapple genome has one fewer ancient whole genome duplications than sequenced grass genomes and, therefore, provides an important reference for elucidating gene content and structure in the last common ancestor of extant members of the grass family (Poaceae). Pineapple has a conserved karyotype with seven pre rho duplication chromosomes that are ancestral to extant grass karyotypes. The pineapple lineage has transitioned from C3 photosynthesis to CAM with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues using beta-carbonic anhydrase (βCA) for initial capture of CO2. Promoter regions of all three βCA genes contain a CCA1 binding site that can bind circadian core oscillators. CAM pathway genes were enriched with cis-regulatory elements including the morning (CCACAC) and evening (AAAATATC) elements associated with regulation of circadian-clock genes, providing the first link between CAM and the circadian clock regulation. Gene-interaction network analysis revealed both activation and repression of regulatory elements that control key enzymes in CAM photosynthesis, indicating that CAM evolved by reconfiguration of pathways preexisting in C3 plants. Pineapple CAM photosynthesis is the result of regulatory neofunctionalization of preexisting gene copies and not acquisition of neofunctionalized genes via whole genome or tandem gene duplication. PMID:26523774

Wellhead treatment costs for groundwater contaminated with pesticides: A preliminary analysis for pineapple in Hawaii

NASA Astrophysics Data System (ADS)

Leon-Guerrero, Ephraim D.; Loague, Keith; Green, Richard E.

1994-01-01

In Hawaii, trace concentrations of pesticides used in the production of pineapple were found in the groundwater supplies of Mililani Town in the Pearl Harbor Basin on the island of Oahu. Groundwater serves as the major source of drinking water and residents pay for wellhead treatment of the contaminated water, via their monthly water bill. The agricultural chemical users within the Pearl Harbor Basin do not include these wellhead treatment costs in their production costs. The agricultural industry benefits from using pesticides but does not pay the entire societal cost of using these chemicals. In this study we evaluate the specific financial cost of wellhead treatment, and not the economic value of groundwater. While wellhead treatment costs could conceivably be shared by several parties, this study focuses on the financial impact of the pineapple industry alone. This study factors annual wellhead treatment costs into annual pineapple production costs to measure the effect on annual financial return from pineapple production. Wellhead treatment costs are calculated from the existing granulated activated carbon (GAC) water treatment facility for Millilani Wells I and II. Pineapple production costs are estimated from previous cost of production studies. The inclusion of wellhead treatment costs produces different production-cost results, depending on the scale of analysis. At the local scale, the Mililani wellhead treatment costs can be factored into the production costs of the pineapple fields, which were probably responsible for contamination of the Mililani Wells, without causing a deficit in economic return. At the larger regional scale, however, the return from all of the pineapple grown in the Pearl Harbor Basin can not sustain the cost of wellhead treatmentfor the entire water supply of the basin. Recommendations point to the prevention of groundwater contamination as more cost-effective measure than wellhead treatment.

Dietary Supplementation with Fresh Pineapple Juice Decreases Inflammation and Colonic Neoplasia in IL-10-deficient Mice with Colitis

PubMed Central

Hale, Laura P.; Chichlowski, Maciej; Trinh, Chau T.; Greer, Paula K.

2010-01-01

Background Bromelain, a mixture of proteolytic enzymes typically derived from pineapple stem, decreases production of pro-inflammatory cytokines and leukocyte homing to sites of inflammation. We previously showed that short-term oral treatment with bromelain purified from pineapple stem decreased the severity of colonic inflammation in C57BL/6 Il10−/− mice with chronic colitis. Since fresh pineapple fruit contains similar bromelain enzymes but at different proportions, this study aimed to determine whether long-term dietary supplementation with pineapple (supplied as juice) could decrease colon inflammation and neoplasia in Il10−/− mice with chronic colitis as compared with bromelain derived from stem. Results Experimental mice readily consumed fresh pineapple juice at a level that generated mean stool proteolytic activities equivalent to 16 mg bromelain purified from stem, while control mice received boiled juice with inactive enzymes. Survival was increased in the group supplemented with fresh rather than boiled juice (p = 0.01). Mice that received fresh juice also had decreased histologic colon inflammation scores and a lower incidence of inflammation-associated colonic neoplasia (35% vs. 66%; p< 0.02), with fewer neoplastic lesions/colon (p = 0.05). Flow cytometric analysis of murine splenocytes exposed to fresh pineapple juice in vitro demonstrated proteolytic removal of cell surface molecules that can affect leukocyte trafficking and activation. Conclusions These results demonstrate that long-term dietary supplementation with fresh or unpasteurized frozen pineapple juice with proteolytically active bromelain enzymes is safe and decreases inflammation severity and the incidence and multiplicity of inflammation-associated colonic neoplasia in this commonly used murine model of inflammatory bowel disease. PMID:20848493

A comparative study of the effects of bromelain and fresh pineapple juice on the early phase of healing in acute crush achilles tendon injury.

PubMed

Aiyegbusi, Ayoola I; Olabiyi, Olaleye O; Duru, Francis I O; Noronha, Cressie C; Okanlawon, Abayomi O

2011-04-01

Bromelain, an enzyme extracted from the stem of the pineapple plant, has been reported to reduce pain and swelling in acute soft tissue injuries, but no study has been done to compare its effect with that of fresh pineapple juice on the healing of acute tendon injuries. This study compared the effects of commercial bromelain and fresh pineapple juice on tenocyte proliferation and the malondialdehyde (MDA) level in the early stage of healing in a crush injury to the Achilles tendon of Sprague-Dawley rats. Twenty-four male rats were divided randomly into three groups of eight rats each; all the rats had induced crush injury to the Achilles tendon: Group 1 (control), no treatment; Group 2, oral bromelain treatment at a dosage of 7 mg/kg of body weight daily; and Group 3, fresh diluted pineapple juice at a dosage of 30 mg/kg of body weight. Treatment was given over the first 14 days post-injury. On day 15 post-injury, the animals were sacrificed, and the tendons were excised and processed for histological study and MDA assay. Results show a significant difference in the tenocyte population between the bromelain group and the control (P < .05), whereas pineapple juice also increased the tenocyte population, although not significantly (P = .36). Pineapple juice, however, significantly lowered the MDA level compared with both the control and bromelain-treated groups. Based on this study, 600 GDU bromelain given at a dosage of 7 mg/kg had a better effect on tenocyte proliferation than fresh pineapple juice given once daily in acute tendon injury.

Residual Expression of the Reprogramming Factors Prevents Differentiation of iPSC Generated from Human Fibroblasts and Cord Blood CD34+ Progenitors

PubMed Central

Ramos-Mejía, Verónica; Montes, Rosa; Bueno, Clara; Ayllón, Verónica; Real, Pedro J.; Rodríguez, René; Menendez, Pablo

2012-01-01

Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation. PMID:22545141

Study of Tensile Properties and Deflection Temperature of Polypropylene/Subang Pineapple Leaf Fiber Composites

NASA Astrophysics Data System (ADS)

Hafizhah, R.; Juwono, A. L.; Roseno, S.

2017-05-01

The development of eco-friendly composites has been increasing in the past four decades because the requirement of eco-friendly materials has been increasing. Indonesia has a lot of natural fiber resources and, pineapple leaf fiber is one of those fibers. This study aimed to determine the influence of weight fraction of pineapple leaf fibers, that were grown at Subang, to the tensile properties and the deflection temperature of polypropylene/Subang pineapple leaf fiber composites. Pineapple leaf fibers were pretreated by alkalization, while polypropylene pellets, as the matrix, were extruded into sheets. Hot press method was used to fabricate the composites. The results of the tensile test and Heat Deflection Temperature (HDT) test showed that the composites that contained of 30 wt.% pineapple leaf fiber was the best composite. The values of tensile strength, modulus of elasticity and deflection temperature were (64.04 ± 3.91) MPa; (3.98 ± 0.55) GPa and (156.05 ± 1.77) °C respectively, in which increased 187.36%, 198.60%, 264.72% respectively from the pristine polypropylene. The results of the observation on the fracture surfaces showed that the failure modes were fiber breakage and matrix failure.

The Mechanical Properties and Microstructure Characters of Hybrid Composite Geopolymers-Pineapple Fiber Leaves (PFL)

NASA Astrophysics Data System (ADS)

Amalia, N.; Hidayatullah, S.; Nurfadilla; Subaer

2017-03-01

The objective of this research is to study the influence of organic fibers on the mechanical properties and microstructure characters of hybrid composite geopolymers-pineapple fibers (PFL). Geopolymers were synthesized by using alkali activated of class C-fly ash added manually with short pineapple fiber leaves (PFL) and then cured at 60°C for 1 hour. The resulting composites were stored in open air for 28 days prior to mechanical and microstructure characterizations. The samples were subjected to compressive and flexural strength measurements, heat resistance as well as acid attack (1M H2SO4 solution). The microstructure of the composites were examined by using Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM-EDS). The measurement showed that the addition of pineapple fibers was able to improve the compressive and flexural strength of geopolymers. The resulting hybrid composites were able to resist fire to a maximum temperature of 1500°C. SEM examination showed the presence of good bond between geopolymer matrix and pineapple fibers. It was also found that there were no chemical constituents of geopolymers leached out during acid liquid treatment. It is concluded that hybrid composite geopolymers-pineapple fibers are potential composites for wide range applications.

Effect of cassava starch coating on quality and shelf life of fresh-cut pineapple (Ananas comosus L. Merril cv "Pérola").

PubMed

Bierhals, Vânia S; Chiumarelli, Marcela; Hubinger, Miriam D

2011-01-01

This research studied the influence of treatment with ascorbic acid, citric acid, and calcium lactate dipping and cassava starch edible coatings on quality parameters and shelf life of fresh-cut pineapple in slices during 12 d at 5 °C. After previous tests, the treatments selected for this study were samples dipped into antibrowning solution with 0.5% of ascorbic acid and 1% of citric acid, with and without 2% of calcium lactate and coated with 2% of cassava starch suspensions. Changes in weight loss, juice leakage, mechanical properties (stress at failure), color parameters (L* and H*), ascorbic acid content, sensory acceptance, and microbial growth of fruits were evaluated. Samples only treated with antibrowning agents were used as control. Edible coatings with and without calcium lactate were efficient in reducing weight loss, juice leakage, and maintaining firmness during storage. However, these samples showed more browning and the ascorbic acid content was reduced. All treatments presented good sensory acceptance (scores above 6). The determining factor of shelf life of pineapple slices was the microbial spoilage. A shelf life of 8 d was obtained for pineapple slices only treated with antibrowning agents. On the other hand, coated samples showed a reduced shelf life of 7 d and higher yeast and mold growth. Thus, although cassava starch coatings were efficient in reducing respiration rate, weight loss, and juice leakage and maintained mechanical properties, these treatments were not able to increase the shelf life of minimally processed pineapple. Practical Application: Pineapple fruit is highly appreciated for its aroma, flavor, and juiciness, but its immediate consumption is difficult. Therefore, pineapple is a potential fruit for minimal processing. However, shelf life of fresh-cut pineapple is very limited by changes in color, texture, appearance, off-flavors, and microbial growth. The use of edible coatings as gas and water vapor barrier and antibrowning agents can extend the storage time and maintain the quality of fresh-cut produce. Cassava starch and alginate coatings are alternative to preserve minimally processed pineapples without changing the quality parameters of fresh fruit. Thus, this study is useful for consumers and fresh-cut industry interested in knowing factors affecting shelf life and quality of fresh-cut pineapple.

Cloning and characterization of the promoter of the 9-cis-epoxycarotenoid dioxygenase gene in Arachis hypogaea L.

PubMed

Liang, Jianhua; Yang, Lixia; Chen, Xiong; Li, Ling; Guo, Dongliang; Li, Haihang; Zhang, Biyu

2009-09-01

We cloned the promoter of the 9-cis-epoxycarotenoid dioxygenase gene from Arachis hypogaea L. beta-Glucuronidase (GUS) histochemical staining and GUS activity assay indicated that the activity of the promoter was exhibited predominantly in the leaves and enhanced by water and NaCl stresses, and by application of abscisic acid (ABA) and salicylic acid (SA) in transgenic Arabidopsis. Moreover, two novel ABRE-like (abscisic acid response element) elements were identified in the promoter region.

Genetic Diversity in Various Accessions of Pineapple [Ananas comosus (L.) Merr.] Using ISSR and SSR Markers.

PubMed

Wang, Jian-Sheng; He, Jun-Hu; Chen, Hua-Rui; Chen, Ye-Yuan; Qiao, Fei

2017-12-01

Inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers were used to assess the genetic diversity of 36 pineapple accessions that were introduced from 10 countries/regions. Thirteen ISSR primers amplified 96 bands, of which 91 (93.65%) were polymorphic, whereas 20 SSR primers amplified 73 bands, of which 70 (96.50%) were polymorphic. Nei's gene diversity (h = 0.28), Shannon's information index (I = 0.43), and polymorphism information content (PIC = 0.29) generated using the SSR primers were higher than that with ISSR primers (h =  0.23, I = 0.37, PIC = 0.24), thereby suggesting that the SSR system is more efficient than the ISSR system in assessing genetic diversity in various pineapple accessions. Mean genetic similarities were 0.74, 0.61, and 0.69, as determined using ISSR, SSR, and combined ISSR/SSR, respectively. These results suggest that the genetic diversity among pineapple accessions is very high. We clustered the 36 pineapple accessions into three or five groups on the basis of the phylogenetic trees constructed based on the results of ISSR, SSR, and combined ISSR/SSR analyses using the unweighted pair-group with arithmetic averaging (UPGMA) method. The results of principal components analysis (PCA) also supported the UPGMA clustering. These results will be useful not only for the scientific conservation and management of pineapple germplasm but also for the improvement of the current pineapple breeding strategies.

De novo assembly, characterization and functional annotation of pineapple fruit transcriptome through massively parallel sequencing.

PubMed

Ong, Wen Dee; Voo, Lok-Yung Christopher; Kumar, Vijay Subbiah

2012-01-01

Pineapple (Ananas comosus var. comosus), is an important tropical non-climacteric fruit with high commercial potential. Understanding the mechanism and processes underlying fruit ripening would enable scientists to enhance the improvement of quality traits such as, flavor, texture, appearance and fruit sweetness. Although, the pineapple is an important fruit, there is insufficient transcriptomic or genomic information that is available in public databases. Application of high throughput transcriptome sequencing to profile the pineapple fruit transcripts is therefore needed. To facilitate this, we have performed transcriptome sequencing of ripe yellow pineapple fruit flesh using Illumina technology. About 4.7 millions Illumina paired-end reads were generated and assembled using the Velvet de novo assembler. The assembly produced 28,728 unique transcripts with a mean length of approximately 200 bp. Sequence similarity search against non-redundant NCBI database identified a total of 16,932 unique transcripts (58.93%) with significant hits. Out of these, 15,507 unique transcripts were assigned to gene ontology terms. Functional annotation against Kyoto Encyclopedia of Genes and Genomes pathway database identified 13,598 unique transcripts (47.33%) which were mapped to 126 pathways. The assembly revealed many transcripts that were previously unknown. The unique transcripts derived from this work have rapidly increased of the number of the pineapple fruit mRNA transcripts as it is now available in public databases. This information can be further utilized in gene expression, genomics and other functional genomics studies in pineapple.

De Novo Assembly, Characterization and Functional Annotation of Pineapple Fruit Transcriptome through Massively Parallel Sequencing

PubMed Central

Ong, Wen Dee; Voo, Lok-Yung Christopher; Kumar, Vijay Subbiah

2012-01-01

Background Pineapple (Ananas comosus var. comosus), is an important tropical non-climacteric fruit with high commercial potential. Understanding the mechanism and processes underlying fruit ripening would enable scientists to enhance the improvement of quality traits such as, flavor, texture, appearance and fruit sweetness. Although, the pineapple is an important fruit, there is insufficient transcriptomic or genomic information that is available in public databases. Application of high throughput transcriptome sequencing to profile the pineapple fruit transcripts is therefore needed. Methodology/Principal Findings To facilitate this, we have performed transcriptome sequencing of ripe yellow pineapple fruit flesh using Illumina technology. About 4.7 millions Illumina paired-end reads were generated and assembled using the Velvet de novo assembler. The assembly produced 28,728 unique transcripts with a mean length of approximately 200 bp. Sequence similarity search against non-redundant NCBI database identified a total of 16,932 unique transcripts (58.93%) with significant hits. Out of these, 15,507 unique transcripts were assigned to gene ontology terms. Functional annotation against Kyoto Encyclopedia of Genes and Genomes pathway database identified 13,598 unique transcripts (47.33%) which were mapped to 126 pathways. The assembly revealed many transcripts that were previously unknown. Conclusions The unique transcripts derived from this work have rapidly increased of the number of the pineapple fruit mRNA transcripts as it is now available in public databases. This information can be further utilized in gene expression, genomics and other functional genomics studies in pineapple. PMID:23091603

Production of recombinant albumin by a herd of cloned transgenic cattle.

PubMed

Echelard, Yann; Williams, Jennifer L; Destrempes, Margaret M; Koster, Julie A; Overton, Susan A; Pollock, Daniel P; Rapiejko, Karen T; Behboodi, Esmail; Masiello, Nicholas C; Gavin, William G; Pommer, Jerry; Van Patten, Scott M; Faber, David C; Cibelli, Jose B; Meade, Harry M

2009-06-01

Purified plasma derived human albumin has been available as a therapeutic product since World War II. However, cost effective recombinant production of albumin has been challenging due to the amount needed and the complex folding pattern of the protein. In an effort to provide an abundant source of recombinant albumin, a herd of transgenic cows expressing high levels of rhA in their milk was generated. Expression cassettes efficiently targeting the secretion of human albumin to the lactating mammary gland were obtained and tested in transgenic mice. A high expressing transgene was transfected in primary bovine cell lines to produce karyoplasts for use in a somatic cell nuclear transfer program. Founder transgenic cows were produced from four independent cell lines. Expression levels varying from 1-2 g/l to more than 40 g/l of correctly folded albumin were observed. The animals expressing the highest levels of rhA exhibited shortened lactation whereas cows yielding 1-2 g/l had normal milk production. This herd of transgenic cattle is an easily scalable and well characterized source of rhA for biomedical uses.

Expression and characterization of bioactive recombinant human alpha-lactalbumin in the milk of transgenic cloned cows.

PubMed

Wang, J; Yang, P; Tang, B; Sun, X; Zhang, R; Guo, C; Gong, G; Liu, Y; Li, R; Zhang, L; Dai, Y; Li, N

2008-12-01

Improvement of the nutritional value of cow milk with transgenic expression of recombinant human alpha-lactalbumin (alpha-LA) has been previously attempted. However, the detailed characterization of the recombinant protein and analysis of the transgenic milk components are not explored yet. Here, we first report production of healthy transgenic cows by somatic cell nuclear transfer, in which expression of up to 1.55 g/L of recombinant human alpha-LA was achieved. The recombinant human alpha-LA was purified from transgenic milk and displayed physicochemical properties similar to its natural counterpart with respect to molecular weight, structure, and regulatory activity for beta-1,4-galactosyltransferase. Additionally, no N-glycosylation was found in the recombinant human alpha-LA, whereas the endogenous bovine alpha-LA was glycosylated at the unusual site (71)Asn-Ile-(73)Cys. Compared with milk from nontransgenic cows, expression of the transgene did not materially alter milk composition, such as fat and protein content. Our research thus provides scientific evidence supporting the feasibility of humanizing cow milk.

Introgression of the SbASR-1 Gene Cloned from a Halophyte Salicornia brachiata Enhances Salinity and Drought Endurance in Transgenic Groundnut (Arachis hypogaea) and Acts as a Transcription Factor

PubMed Central

Tiwari, Vivekanand; Chaturvedi, Amit Kumar; Mishra, Avinash; Jha, Bhavanath

2015-01-01

The SbASR-1 gene, cloned from a halophyte Salicornia brachiata, encodes a plant-specific hydrophilic and stress responsive protein. The genome of S. brachiata has two paralogs of the SbASR-1 gene (2549 bp), which is comprised of a single intron of 1611 bp, the largest intron of the  abscisic acid stress ripening [ASR] gene family yet reported. In silico analysis of the 843-bp putative promoter revealed the presence of ABA, biotic stress, dehydration, phytohormone, salinity, and sugar responsive cis-regulatory motifs. The SbASR-1 protein belongs to Group 7 LEA protein family with different amino acid composition compared to their glycophytic homologs. Bipartite Nuclear Localization Signal (NLS) was found on the C-terminal end of protein and localization study confirmed that SbASR-1 is a nuclear protein. Furthermore, transgenic groundnut (Arachis hypogaea) plants over-expressing the SbASR-1 gene constitutively showed enhanced salinity and drought stress tolerance in the T1 generation. Leaves of transgenic lines exhibited higher chlorophyll and relative water contents and lower electrolyte leakage, malondialdehyde content, proline, sugars, and starch accumulation under stress treatments than wild-type (Wt) plants. Also, lower accumulation of H2O2 and O2 .- radicals was detected in transgenic lines compared to Wt plants under stress conditions. Transcript expression of APX (ascorbate peroxidase) and CAT (catalase) genes were higher in Wt plants, whereas the SOD (superoxide dismutase) transcripts were higher in transgenic lines under stress. Electrophoretic mobility shift assay (EMSA) confirmed that the SbASR-1 protein binds at the consensus sequence (C/G/A)(G/T)CC(C/G)(C/G/A)(A/T). Based on results of the present study, it may be concluded that SbASR-1 enhances the salinity and drought stress tolerance in transgenic groundnut by functioning as a LEA (late embryogenesis abundant) protein and a transcription factor. PMID:26158616

Sharing Malaysian experience with the development of biotechnology-derived food crops.

PubMed

Abu Bakar, Umi K; Pillai, Vilasini; Hashim, Marzukhi; Daud, Hassan Mat

2005-12-01

Biotechnology-derived food crops are currently being developed in Malaysia mainly for disease resistance and improved post harvest quality. The modern biotechnology approach is adopted because of its potential to overcome constraints faced by conventional breeding techniques. Research on the development of biotechnology-derived papaya, pineapple, chili, passion fruit, and citrus is currently under way. Biotechnology-derived papaya developed for resistance to papaya ringspot virus (PRSV) and improved postharvest qualities is at the field evaluation stage. Pineapple developed for resistance to fruit black heart disorder is also being evaluated for proof-of-concept. Other biotechnology-derived food crops are at early stages of gene cloning and transformation. Activities and products involving biotechnology-derived crops will be fully regulated in the near future under the Malaysian Biosafety Law. At present they are governed only by guidelines formulated by the Genetic Modification Advisory Committee (GMAC), Malaysia. Commercialization of biotechnology-derived crops involves steps that require GMAC approval for all field evaluations and food-safety assessments before the products are placed on the market. Public acceptance of the biotechnology product is another important factor for successful commercialization. Understanding of biotechnology is generally low among Malaysians, which may lead to low acceptance of biotechnology-derived products. Initiatives are being taken by local organizations to improve public awareness and acceptance of biotechnology. Future research on plant biotechnology will focus on the development of nutritionally enhanced biotechnology-derived food crops that can provide more benefits to consumers.

A Modular Toolset for Recombination Transgenesis and Neurogenetic Analysis of Drosophila

PubMed Central

Wang, Ji-Wu; Beck, Erin S.; McCabe, Brian D.

2012-01-01

Transgenic Drosophila have contributed extensively to our understanding of nervous system development, physiology and behavior in addition to being valuable models of human neurological disease. Here, we have generated a novel series of modular transgenic vectors designed to optimize and accelerate the production and analysis of transgenes in Drosophila. We constructed a novel vector backbone, pBID, that allows both phiC31 targeted transgene integration and incorporates insulator sequences to ensure specific and uniform transgene expression. Upon this framework, we have built a series of constructs that are either backwards compatible with existing restriction enzyme based vectors or utilize Gateway recombination technology for high-throughput cloning. These vectors allow for endogenous promoter or Gal4 targeted expression of transgenic proteins with or without fluorescent protein or epitope tags. In addition, we have generated constructs that facilitate transgenic splice isoform specific RNA inhibition of gene expression. We demonstrate the utility of these constructs to analyze proteins involved in nervous system development, physiology and neurodegenerative disease. We expect that these reagents will facilitate the proficiency and sophistication of Drosophila genetic analysis in both the nervous system and other tissues. PMID:22848718

Map-based cloning of a gene controlling Omega-3 fatty acid desaturation in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arondel, V.; Lemieux, B.; Hwang, I.

1992-11-20

A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase. 24 refs., 2 figs., 1more » tabs.« less

Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

Ripening-dependent metabolic changes in the volatiles of pineapple (Ananas comosus (L.) Merr.) fruit: II. Multivariate statistical profiling of pineapple aroma compounds based on comprehensive two-dimensional gas chromatography-mass spectrometry.

PubMed

Steingass, Christof Björn; Jutzi, Manfred; Müller, Jenny; Carle, Reinhold; Schmarr, Hans-Georg

2015-03-01

Ripening-dependent changes of pineapple volatiles were studied in a nontargeted profiling analysis. Volatiles were isolated via headspace solid phase microextraction and analyzed by comprehensive 2D gas chromatography and mass spectrometry (HS-SPME-GC×GC-qMS). Profile patterns presented in the contour plots were evaluated applying image processing techniques and subsequent multivariate statistical data analysis. Statistical methods comprised unsupervised hierarchical cluster analysis (HCA) and principal component analysis (PCA) to classify the samples. Supervised partial least squares discriminant analysis (PLS-DA) and partial least squares (PLS) regression were applied to discriminate different ripening stages and describe the development of volatiles during postharvest storage, respectively. Hereby, substantial chemical markers allowing for class separation were revealed. The workflow permitted the rapid distinction between premature green-ripe pineapples and postharvest-ripened sea-freighted fruits. Volatile profiles of fully ripe air-freighted pineapples were similar to those of green-ripe fruits postharvest ripened for 6 days after simulated sea freight export, after PCA with only two principal components. However, PCA considering also the third principal component allowed differentiation between air-freighted fruits and the four progressing postharvest maturity stages of sea-freighted pineapples.

Cloned transgenic heart-healthy pork?

PubMed

Prather, Randall S

2006-08-01

Here I comment on the production and uses of swine that express a humanized fat-1 gene. The gene product is a fatty acid desaturase that converts omega-6 fatty acids to omega-3 fatty acids. Omega-3 fatty acids have been implicated as being important for reproductive success, maintaining a healthy cardiovascular system, sustaining a functional immune system, and even preventing depression and cancer. The descendants of these hfat-1 transgenic swine will be very useful as models of the human condition, and if they are permitted to enter the food chain, they may improve human health.

The Role of Amplified Wild-Type Neu in the Etiology of Breast Cancer

DTIC Science & Technology

2004-07-01

mutations that were found, only one (a C - T change of chromosome 5 (data not shown). DAPI banding patterns of nucleotide 2137) was present in clones...activation. Proc. NatI. Acad. Sci. USA 93, 8878-8883. Koelsch, B.U. (1998). Assignment of erbB2 to rat chromosome band 1Oq32.1 by in situ...was used to map the integration site of the neu transgene in lines 6500 and 2477. Line 6500 contained -3-6 copies of the transgene in chromosome 10

Quality of osmotically pre-treated and vacuum dried pineapple cubes on storage as influenced by type of solutes and packaging materials.

PubMed

Paul, Prodyut Kumar; Ghosh, Swapan Kumar; Singh, Dhananjay Kumar; Bhowmick, Nilesh

2014-08-01

The quality and stability of osmotically pre-treated and subsequently vacuum dried pineapple cubes using three different solutes and packed in three different types of packaging materials on storage was evaluated. The experiment was laid out in completely randomized block design with two factors and three replications for each treatment. Treatment combinations were considered as one factor and storage interval as another factor. Pineapple cubes stored in glass bottle showed very little percentage variation in moisture content due to its high moisture barrier properties. In all treatment combination, acidity values were invariably found to increase as the storage progressed. For all three different osmotic treatments, HDPE pouch packet always showed highest acidity followed by PVDC pouch. Again among three solutes under consideration, invert sugar recorded a rapid increase in acidity than other solutes. In pineapple cubes osmotically treated with sucrose solution, the rates of decrease of total sugar content were lower than that of invert sugar and sorbitol treated pineapple cubes. The percentage decrease of total sugar content was highest when the osmotically dehydrated pineapple cubes were packed in HDPE pouch and it was least in glass bottles. There was a gradual decrease in ascorbic acid content with the extension of storage period and this decrease was statistically significant at all storage intervals up to six-month. Lowest value of ascorbic acid content (15.210 mg per 100 g initial solid) was recorded in invert sugar treated pineapple cube packed in HDPE pouch after 6 months of storage.

Diurnal Cycling Transcription Factors of Pineapple Revealed by Genome-Wide Annotation and Global Transcriptomic Analysis

PubMed Central

Sharma, Anupma; Wai, Ching Man; Ming, Ray

2017-01-01

Abstract Circadian clock provides fitness advantage by coordinating internal metabolic and physiological processes to external cyclic environments. Core clock components exhibit daily rhythmic changes in gene expression, and the majority of them are transcription factors (TFs) and transcription coregulators (TCs). We annotated 1,398 TFs from 67 TF families and 80 TCs from 20 TC families in pineapple, and analyzed their tissue-specific and diurnal expression patterns. Approximately 42% of TFs and 45% of TCs displayed diel rhythmic expression, including 177 TF/TCs cycling only in the nonphotosynthetic leaf tissue, 247 cycling only in the photosynthetic leaf tissue, and 201 cycling in both. We identified 68 TF/TCs whose cycling expression was tightly coupled between the photosynthetic and nonphotosynthetic leaf tissues. These TF/TCs likely coordinate key biological processes in pineapple as we demonstrated that this group is enriched in homologous genes that form the core circadian clock in Arabidopsis and includes a STOP1 homolog. Two lines of evidence support the important role of the STOP1 homolog in regulating CAM photosynthesis in pineapple. First, STOP1 responds to acidic pH and regulates a malate channel in multiple plant species. Second, the cycling expression pattern of the pineapple STOP1 and the diurnal pattern of malate accumulation in pineapple leaf are correlated. We further examined duplicate-gene retention and loss in major known circadian genes and refined their evolutionary relationships between pineapple and other plants. Significant variations in duplicate-gene retention and loss were observed for most clock genes in both monocots and dicots. PMID:28922793

Effects of different agricultural systems on soil quality in Northern Limón province, Costa Rica.

PubMed

Cornwell, Emma

2014-09-01

Conversion of native rainforest ecosystems in Limón Province of Costa Rica to banana and pineapple monoculture has led to reductions in biodiversity and soil quality. Agroforestry management of cacao (Theobroma cacao) is an alternative system that may maintain the agricultural livelihood of the region while more closely mimicking native ecosystems. This study compared physical, biological and chemical soil quality indicators of a cacao plantation under organic agroforestry management with banana, pineapple, and pasture systems; a native forest nearby served as a control. For bulk density and earthworm analysis, 18 samples were collected between March and April 2012 from each ecosystem paired with 18 samples from the cacao. Cacao had a lower bulk density than banana and pineapple monocultures, but greater than the forest (p < 0.05). Cacao also hosted a greater number and mass of earthworms than banana and pineapple (p < 0.05), but similar to forest and pasture. For soil chemical characteristics, three composite samples were collected in March 2012 from each agroecosystem paired with three samples from the cacao plantation. Forest and pineapple ecosystems had the lowest pH, cation exchange capacity, and exchangeable nutrient cations, while cacao had the greatest (p < 0.05). Total nutrient levels of P and N were slightly greater in banana, pineapple and pasture than in cacao; probably related to addition of chemical fertilizer and manure from cattle grazing. Forest and cacao also had greater %C, than other ecosystems, which is directly related to soil organic matter content (p < 0.0001). Overall, cacao had more favorable physical, biological and chemical soil characteristics than banana and pineapple monocultures, while trends were less conclusive compared to the pastureland. While organic cacao was inferior to native forest in some soil characteristics such as bulk density and organic carbon, its soil quality did best mimic that of the native forest. This supports the organic cultivation of cacao as a desirable alternative to banana and pineapple monoculture.

Early histological, hormonal, and molecular changes during pineapple (Ananas comosus (L.) Merrill) artificial flowering induction.

PubMed

Espinosa, Maita Eulalia Ávila; Moreira, Rafael Oliveira; Lima, André Almeida; Ságio, Solange Aparecida; Barreto, Horllys Gomes; Luiz, Sara Lazara Pérez; Abreu, Carlos Eduardo Aragón; Yanes-Paz, Ermis; Ruíz, Yanelis Capdesuñer; González-Olmedo, Justo Lorenzo; Chalfun-Júnior, Antonio

2017-02-01

Natural flowering can cause serious scheduling problems in the pineapple (Ananas comosus) industry and increase harvest costs. Pineapple flowering is thought to be triggered by increased ethylene levels and artificial forcing of pineapple flowering is a common practice to promote flowering synchronisation. However, little is known about the early hormonal and molecular changes of pineapple flowering induction and development. Here, we aimed to analyse the molecular, hormonal, and histological changes during artificial pineapple flowering by Ethrel ® 48 treatment. Histological analyses of the shoot apical meristem, leaf gibberellic acid (GA 3 ), and ethylene quantification were carried out during the first 72h after Ethrel ® 48 treatment. Expression profiles from ethylene biosynthesis (AcACS2 and AcACO1), gibberellin metabolism (AcGA2-ox1 and AcDELLA1), and flower development (FT-like gene (AcFT), LFY-like gene (AcLFY), and a PISTILLATA-like gene (AcPI)) genes were analysed during the first 24h after Ethrel ® 48 treatment. Differentiation processes of the shoot apical meristem into flower buds were already present in the first 72h after Ethrel ® 48 treatment. Ethrel ® 48 lead to a reduction in GA 3 levels, probably triggered by elevated ethylene levels and the positive regulation AcGA2-ox1. AcLFY activation upon Ethrel ® 48 may also have contributed to the reduction of GA 3 levels and, along with the up-regulation of AcPI, are probably associated with the flower induction activation. AcFT and AcDELLA1 do not seem to be regulated by GA 3 and ethylene. Decreased GA 3 and increased ethylene levels suggest an accumulation of AcDELLA1, which may display an important role in pineapple flowering induction. Thus, this study shows that molecular, hormonal, and histological changes are present right after Ethrel ® 48 treatment, providing new insights into how pineapple flowering occurs under natural conditions. Copyright © 2016 Elsevier GmbH. All rights reserved.

The involvement of mitochondrial phosphate transporter in accelerating bud dormancy release during chilling treatment of tree peony (Paeonia suffruticosa).

PubMed

Huang, Xin; Zhu, Wei; Dai, Silan; Gai, Shupeng; Zheng, Guosheng; Zheng, Chengchao

2008-09-01

A cDNA clone was isolated from tree peony (Paeonia suffruticosa) subtractive cDNA library of burst buds and characterized with regard to its sequence, expression in response to chilling treatment during the release of bud dormancy, and its function in transgenic Arabidopsis thaliana. The clone, designated as PsMPT, contains 1,615 nucleotides with an open reading frame of 1,119 nucleotides, and the deduced amino acid sequence shows high homology with mitochondrial phosphate transporters (MPTs) from various organisms. The mRNA accumulation of PsMPT in tree peony was strongly induced by chilling treatment during the release of bud dormancy. When the treated plants were transferred to normal growth conditions, the level of PsMPT transcripts induced by sufficient chilling could be maintained high, whereas that induced by insufficient chilling decreased sharply. The transgenic Arabidopsis plants that overexpress PsMPT showed rapid growth and earlier flowering than wild-type plants. ATP contents in the transgenic plants were much higher than that in wild-type plants through various developmental stages. Together, these results suggest that the product of PsMPT is a MPT and might play an important role during the release of bud dormancy in tree peony.

Production and Quality Evaluation of Pineapple Fruit Wine

NASA Astrophysics Data System (ADS)

Qi, Ningli; Ma, Lina; Li, Liuji; Gong, Xiao; Ye, Jianzhi

2017-12-01

The fermentation process of pineapple fruit wine was studied. The juice was inoculated with 5% (v/v) active yeast and held at 20 °C for 7 days. Total sugar and pH decreased while the alcoholic strength increased with increasing length of fermentation. The fermented fruit wine contains 2.29 g/L total acid, 10.2 % (v/v) alcohol, 5.4 °Brix soluble solids, pH 3.52. Pineapple wine detected 68 kinds of aroma components, including 34 esters, 13 alcohols. The ester material accounted for 52.25% of the main aroma components. The quality and sensory evaluation results indicated that pineapple fruit wine belongs to a kind of low alcohol wine, so it is easy to be accepted by the public.

Animal breeding in the age of biotechnology: the investigative pathway behind the cloning of Dolly the sheep.

PubMed

García-Sancho, Miguel

2015-09-01

This paper addresses the 1996 cloning of Dolly the sheep, locating it within a long-standing tradition of animal breeding research in Edinburgh. Far from being an end in itself, the cell-nuclear transfer experiment from which Dolly was born should be seen as a step in an investigative pathway that sought the production of medically relevant transgenic animals. By historicising Dolly, I illustrate how the birth of this sheep captures a dramatic redefinition of the life sciences, when in the 1970s and 1980s the rise of neo-liberal governments and the emergence of the biotechnology market pushed research institutions to show tangible applications of their work. Through this broader interpretative framework, the Dolly story emerges as a case study of the deep transformations of agricultural experimentation during the last third of the twentieth century. The reorganisation of laboratory practice, human resources and institutional settings required by the production of transgenic animals had unanticipated consequences. One of these unanticipated effects was that the boundaries between animal and human health became blurred. As a result of this, new professional spaces emerged and the identity of Dolly the sheep was reconfigured, from an instrument for livestock improvement in the farm to a more universal symbol of the new cloning age.

Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis.

PubMed

Tanaka, Toru; Hosoi, Fumihito; Yamaguchi-Iwai, Yuko; Nakamura, Hajime; Masutani, Hiroshi; Ueda, Shugo; Nishiyama, Akira; Takeda, Shunichi; Wada, Hiromi; Spyrou, Giannis; Yodoi, Junji

2002-04-02

Thioredoxin-2 (Trx-2) is a mitochondria-specific member of the thioredoxin superfamily. Mitochondria have a crucial role in the signal transduction for apoptosis. To investigate the biological significance of Trx-2, we cloned chicken TRX-2 cDNA and generated clones of the conditional Trx-2-deficient cells using chicken B-cell line, DT40. Here we show that TRX-2 is an essential gene and that Trx-2-deficient cells undergo apoptosis upon repression of the TRX-2 transgene, showing an accumulation of intracellular reactive oxygen species (ROS). Cytochrome c is released from mitochondria, while caspase-9 and caspase-3, but not caspase-8, are activated upon inhibition of the TRX-2 transgene. In addition, Trx-2 and cytochrome c are co-immunoprecipitated in an in vitro assay. These results suggest that mitochondrial Trx-2 is essential for cell viability, playing a crucial role in the scavenging ROS in mitochondria and regulating the mitochondrial apoptosis signaling pathway.

Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

NASA Astrophysics Data System (ADS)

Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

2009-09-01

Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

Molecular cloning and characterization of the MsHSP17.7 gene from Medicago sativa L.

PubMed

Li, Zhen-Yi; Long, Rui-Cai; Zhang, Tie-Jun; Yang, Qing-Chuan; Kang, Jun-Mei

2016-08-01

Heat shock proteins (HSPs) are ubiquitous protective proteins that play crucial roles in plant development and adaptation to stress, and the aim of this study is to characterize the HSP gene in alfalfa. Here we isolated a small heat shock protein gene (MsHSP17.7) from alfalfa by homology-based cloning. MsHSP17.7 contains a 477-bp open reading frame and encodes a protein of 17.70-kDa. The amino acid sequence shares high identity with MtHSP (93.98 %), PsHSP17.1 (83.13 %), GmHSP17.9 (74.10 %) and SlHSP17.6 (79.25 %). Phylogenetic analysis revealed that MsHSP17.7 belongs to the group of cytosolic class II small heat shock proteins (sHSP), and likely localizes to the cytoplasm. Quantitative RT-PCR indicated that MsHSP17.7 was induced by heat shock, high salinity, peroxide and drought stress. Prokaryotic expression indicated that the salt and peroxide tolerance of Escherichia coli was remarkably enhanced. Transgenic Arabidopsis plants overexpressing MsHSP17.7 exhibited increased root length of transgenic Arabidopsis lines under salt stress compared to the wild-type line. The malondialdehyde (MDA) levels in the transgenic lines were significantly lower than in wild-type, although proline levels were similar between transgenic and wild-type lines. MsHSP17.7 was induced by heat shock, high salinity, oxidative stress and drought stress. Overexpression analysis suggests that MsHSP17.7 might play a key role in response to high salinity stress.

On-line gas chromatography combustion/pyrolysis isotope ratio mass spectrometry (HRGC-C/P-IRMS) of pineapple (Ananas comosus L. Merr.) volatiles.

PubMed

Preston, Christina; Richling, Elke; Elss, Sandra; Appel, Markus; Heckel, Frank; Hartlieb, Ariane; Schreier, Peter

2003-12-31

By use of extracts prepared by liquid-liquid separation of the volatiles from self-prepared juices of pineapple fruits (Ananas comosus) (n = 14) as well as commercial pineapple recovery aromas/water phases (n = 3), on-line capillary gas chromatography-isotope ratio mass spectrometry was employed in the combustion (C) and the pyrolysis (P) modes (HRGC-C/P-IRMS) to determine the delta(13)C(VPDB) and delta(2)H(VSMOW) values of selected pineapple flavor constituents. In addition to methyl 2-methylbutanoate 1, ethyl 2-methylbutanoate 2, methyl hexanoate 3, ethyl hexanoate 4, and 2,5-dimethyl-4-methoxy-3[2H]-furanone 5, each originating from the fruit, the delta(13)C(VPDB) and delta(2)H(VSMOW) data of commercial synthetic 1-5 and "natural" (biotechnologically derived) 1-4 were determined. With delta(13)C(VPDB) data of pineapple volatiles 1-4 varying from -12.8 to -24.4 per thousand, the range expected for CAM metabolism was observed. Compound 5 showed higher depletion from -20.9 to -28.6 per thousand. A similar situation was given for the delta(2)H(VSMOW) values of 3-5 from pineapple ranging from -118 to -191 per thousand, whereas 1 and 2 showed higher depleted values from -184 to -263 per thousand. In nearly all cases, analytical differentiation of 1-5 from pineapple and natural as well as synthetic origin was possible. In general, natural and synthetic 1-5 exhibited delta(13)C(VPDB) data ranging from -11.8 to -32.2 per thousand and -22.7 to -35.9 per thousand, respectively. Their delta(2)H(VSMOW) data were in the range from -242 to -323 per thousand and -49 to -163 per thousand, respectively.

Diurnal Cycling Transcription Factors of Pineapple Revealed by Genome-Wide Annotation and Global Transcriptomic Analysis.

PubMed

Sharma, Anupma; Wai, Ching Man; Ming, Ray; Yu, Qingyi

2017-09-01

Circadian clock provides fitness advantage by coordinating internal metabolic and physiological processes to external cyclic environments. Core clock components exhibit daily rhythmic changes in gene expression, and the majority of them are transcription factors (TFs) and transcription coregulators (TCs). We annotated 1,398 TFs from 67 TF families and 80 TCs from 20 TC families in pineapple, and analyzed their tissue-specific and diurnal expression patterns. Approximately 42% of TFs and 45% of TCs displayed diel rhythmic expression, including 177 TF/TCs cycling only in the nonphotosynthetic leaf tissue, 247 cycling only in the photosynthetic leaf tissue, and 201 cycling in both. We identified 68 TF/TCs whose cycling expression was tightly coupled between the photosynthetic and nonphotosynthetic leaf tissues. These TF/TCs likely coordinate key biological processes in pineapple as we demonstrated that this group is enriched in homologous genes that form the core circadian clock in Arabidopsis and includes a STOP1 homolog. Two lines of evidence support the important role of the STOP1 homolog in regulating CAM photosynthesis in pineapple. First, STOP1 responds to acidic pH and regulates a malate channel in multiple plant species. Second, the cycling expression pattern of the pineapple STOP1 and the diurnal pattern of malate accumulation in pineapple leaf are correlated. We further examined duplicate-gene retention and loss in major known circadian genes and refined their evolutionary relationships between pineapple and other plants. Significant variations in duplicate-gene retention and loss were observed for most clock genes in both monocots and dicots. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Biology and trapping of stable flies (Diptera: Muscidae) developing in pineapple residues (Ananas comosus) in Costa Rica.

PubMed

Solórzano, José-Arturo; Gilles, Jeremie; Bravo, Oscar; Vargas, Cristina; Gomez-Bonilla, Yannery; Bingham, Georgina V; Taylor, David B

2015-01-01

Pineapple production in Costa Rica increased nearly 300-fold during the last 30 yr, and >40,000 hectares of land are currently dedicated to this crop. At the end of the pineapple cropping cycle, plants are chopped and residues incorporated into the soil in preparation for replanting. Associated with increased pineapple production has been a large increase in stable fly, Stomoxys calcitrans (L.), populations. Stable flies are attracted to, and oviposit in, the decomposing, chopped pineapple residues. In conjunction with chemical control of developing larvae, adult trapping is an important control strategy. In this study, four blue-black fabric traps, Nzi, Vavoua, Model H, and Ngu, were compared with a white sticky trap currently used for stable fly control in Costa Rica. Overall, the white sticky trap caught the highest number of stable flies, followed by the Nzi, Vavoua, Model H, and Ngu. Collections on the white sticky trap increased 16 d after residues were chopped; coinciding with the expected emergence of flies developing in the pineapple residues. During this same time period, collections in the blue-black fabric traps decreased. Sex ratio decreased from >7:1 (females:males) 3-7 d after chopping to 1:1 at 24-28 d. White sticky, Nzi and Vavoua traps collected similar numbers of colonizing flies 3-7 d after residues were chopped. However, white sticky traps collected more flies once emergence from the pineapple residues began. Although white sticky traps collected more flies than fabric traps, they remain labor intensive and environmentally unsound because of their disposable and nonbiodegradable nature. Published by Oxford University Press on behalf of the Entomological Society of America 2015. This work is written by US Government employees and is in the public domain in the US.

Understanding Extreme Precipitation Behaviour in British Columbia's Lower Mainland Using Historical and Proxy Records

NASA Astrophysics Data System (ADS)

Spry, Christina

In British Columbia, Pineapple Express storms can lead to flooding, slope failures and negative impacts to water quality. Mitigating the impacts of extreme weather events in a changing climate requires an understanding of how local climate responds to regional-toglobal climate forcing patterns. In this study, I use historical and proxy data to identify the distinguishing characteristics of Pineapple Express storms and to develop a tree ring oxygen isotope record (1960--1995) of local climate conditions in the Lower Mainland of British Columbia. I found that high magnitude Pineapple Express storms have significantly higher precipitation and streamflow than other storms types, which result in relatively high contributions of Pineapple Express storms to the annual water budget. As well, Pineapple Express precipitation is characterized by an enriched delta18O isotopic signature when compared to precipitation originating from the North Pacific Ocean. However, differences in source water do not appear to be driving the variability in tree ring delta18O ratios. Instead, tree ring isotopic values exhibit a regional climate pattern that is strongly driven by latitudinal temperature gradients and the Rayleigh distillation effect. Therefore, future warmer conditions may decrease the temperature gradient between the equator and the poles, which can be recorded in the tree ring isotope record. The results also suggest that warmer temperatures due to climate change could result in more active Pineapple Express storm seasons, with multiple PE storms happening over a short period of time. Concurrent storms significantly increase the risk to society because the resulting antecedent saturated soil conditions can trigger precipitationinduced natural hazards. Keywords: extreme weather; stable isotopes; Pineapple Express; British Columbia; climate change; tree rings.

Inactivation of Escherichia coli, Listeria monocytogenes, and Salmonella Enteritidis by Cymbopogon citratus D.C. Stapf. Essential Oil in Pineapple Juice.

PubMed

Leite, Caroline Junqueira Barcellos; de Sousa, Jossana Pereira; Medeiros, José Alberto da Costa; da Conceição, Maria Lúcia; dos Santos Falcão-Silva, Vivyanne; de Souza, Evandro Leite

2016-02-01

In the present study, the efficacy of Cymbopogon citratus D.C. Stapf. essential oil (CCEO) to provoke a 5-log CFU/ml (5-log) inactivation in a mixed composite of Escherichia coli, Listeria monocytogenes, and Salmonella enterica serovar Enteritidis in pineapple (Ananas comosus (L.) Merril) juice (4°C) was assessed. Moreover, the effects of CCEO on the physicochemical and sensory quality parameters of pineapple juice were evaluated. The MIC of CCEO was 5 μl/ml against the composite mix examined. For L. monocytogenes and E. coli inoculated in juice containing CCEO (5, 2.5, and 1.25 μl/ml), a ≥5-log reduction was detected after 15 min of exposure. This same result was obtained for Salmonella Enteritidis incubated alone in pineapple juice containing CCEO at 5 and 2.5 μl/ml. Overall, Salmonella Enteritidis was the most tolerant and L. monocytogenes was the most sensitive to CCEO. The physicochemical properties (pH, titratable acidic [citric acid per 100 g], and soluble solids) of pineapple juice containing CCEO (2.5 and 1.25 μl/ml) were maintained. Juice containing CCEO (2.5 and 1.25 μl/ml) exhibited similar scores for odor, appearance, and viscosity compared with juice without CCEO. However, unsatisfactory changes in taste and aftertaste were observed in juices containing CCEO. These results suggest that CCEO could be used as an alternative antimicrobial compound to ensure the safety of pineapple juice, although CCEO at the tested concentrations negatively impacted its taste. Therefore, further studies are needed to determine the balance between microbial safety and taste acceptability of pineapple juice containing CCEO.

Biology and Trapping of Stable Flies (Diptera: Muscidae) Developing in Pineapple Residues (Ananas comosus) in Costa Rica

PubMed Central

Solórzano, José-Arturo; Gilles, Jeremie; Bravo, Oscar; Vargas, Cristina; Gomez-Bonilla, Yannery; Bingham, Georgina V.; Taylor, David B.

2015-01-01

Pineapple production in Costa Rica increased nearly 300-fold during the last 30 yr, and >40,000 hectares of land are currently dedicated to this crop. At the end of the pineapple cropping cycle, plants are chopped and residues incorporated into the soil in preparation for replanting. Associated with increased pineapple production has been a large increase in stable fly, Stomoxys calcitrans (L.), populations. Stable flies are attracted to, and oviposit in, the decomposing, chopped pineapple residues. In conjunction with chemical control of developing larvae, adult trapping is an important control strategy. In this study, four blue-black fabric traps, Nzi, Vavoua, Model H, and Ngu, were compared with a white sticky trap currently used for stable fly control in Costa Rica. Overall, the white sticky trap caught the highest number of stable flies, followed by the Nzi, Vavoua, Model H, and Ngu. Collections on the white sticky trap increased 16 d after residues were chopped; coinciding with the expected emergence of flies developing in the pineapple residues. During this same time period, collections in the blue-black fabric traps decreased. Sex ratio decreased from >7:1 (females:males) 3–7 d after chopping to 1:1 at 24–28 d. White sticky, Nzi and Vavoua traps collected similar numbers of colonizing flies 3–7 d after residues were chopped. However, white sticky traps collected more flies once emergence from the pineapple residues began. Although white sticky traps collected more flies than fabric traps, they remain labor intensive and environmentally unsound because of their disposable and nonbiodegradable nature. PMID:26454479

Current status of tropical fruit breeding and genetics for three tropical fruit species cultivated in Japan: pineapple, mango, and papaya

PubMed Central

Ogata, Tatsushi; Yamanaka, Shinsuke; Shoda, Moriyuki; Urasaki, Naoya; Yamamoto, Toshiya

2016-01-01

Tropical fruit crops are predominantly produced in tropical and subtropical developing countries, but some are now grown in southern Japan. Pineapple (Ananas comosus), mango (Mangifera indica) and papaya (Carica papaya) are major tropical fruits cultivated in Japan. Modern, well-organized breeding systems have not yet been developed for most tropical fruit species. Most parts of Japan are in the temperate climate zone, but some southern areas such as the Ryukyu Islands, which stretch from Kyushu to Taiwan, are at the northern limits for tropical fruit production without artificial heating. In this review, we describe the current status of tropical fruit breeding, genetics, genomics, and biotechnology of three main tropical fruits (pineapple, mango, and papaya) that are cultivated and consumed in Japan. More than ten new elite cultivars of pineapple have been released with improved fruit quality and suitability for consumption as fresh fruit. New challenges and perspectives for obtaining high fruit quality are discussed in the context of breeding programs for pineapple. PMID:27069392

Pineapple (Ananas comosus L. Merr.) wine production in Angola: Characterisation of volatile aroma compounds and yeast native flora.

PubMed

Dellacassa, Eduardo; Trenchs, Oriol; Fariña, Laura; Debernardis, Florencia; Perez, Gabriel; Boido, Eduardo; Carrau, Francisco

2017-01-16

A pineapple vinification process was conducted through inoculated and spontaneous fermentation to develop a process suitable for a quality beverage during two successive vintages in Huambo, Angola. Wines obtained with the conventional Saccharomyces cerevisiae strain, were analysed by gas chromatography, and a total of 61 volatile constituents were detected in the volatile fraction and 18 as glycosidically bound aroma compounds. Concentration levels of carbonyl and sulphur compounds were in agreement with the limited information reported about pineapple fruits of other regions. We report, for the first time in pineapple wines, the presence of significant concentrations of lactones, ketones, terpenes, norisoprenoids and a variety of volatile phenols. Eight native yeast strains were isolated from spontaneous batches. Further single-strain fermentations allowed us to characterise their suitability for commercial fermentation. Three native strains (Hanseniaspora opuntiae, H. uvarum and Meyerozyma guilliermondii) were selected with sensory potential to ferment pineapple fruits with increased flavour diversity. Results obtained here contribute to a better understanding of quality fermentation alternatives of this tropical fruit in subtropical regions. Copyright © 2016 Elsevier B.V. All rights reserved.

Transcriptome Profiling of the Pineapple under Low Temperature to Facilitate Its Breeding for Cold Tolerance

PubMed Central

Chen, Chengjie; Zhang, Yafeng; Xu, Zhiqiang; Luan, Aiping; Mao, Qi; Feng, Junting; Xie, Tao; Gong, Xue; Wang, Xiaoshuang; Chen, Hao; He, Yehua

2016-01-01

The pineapple (Ananas comosus) is cold sensitive. Most cultivars are injured during winter periods, especially in sub-tropical regions. There is a lack of molecular information on the pineapple’s response to cold stress. In this study, high-throughput transcriptome sequencing and gene expression analysis were performed on plantlets of a cold-tolerant genotype of the pineapple cultivar ‘Shenwan’ before and after cold treatment. A total of 1,186 candidate cold responsive genes were identified, and their credibility was confirmed by RT-qPCR. Gene set functional enrichment analysis indicated that genes related to cell wall properties, stomatal closure and ABA and ROS signal transduction play important roles in pineapple cold tolerance. In addition, a protein association network of CORs (cold responsive genes) was predicted, which could serve as an entry point to dissect the complex cold response network. Our study found a series of candidate genes and their association network, which will be helpful to cold stress response studies and pineapple breeding for cold tolerance. PMID:27656892

The expression patterns of bromelain and AcCYS1 correlate with blackheart resistance in pineapple fruits submitted to postharvest chilling stress.

PubMed

Raimbault, Astrid-Kim; Zuily-Fodil, Yasmine; Soler, Alain; Mora, Phillipe; Cruz de Carvalho, Maria H

2013-11-01

Blackheart is a physiological disorder induced by postharvest chilling storage during pineapple fruit export shipping. The aim of this study was to check the involvement of bromelain, the cysteine protease protein family abundantly present in pineapple fruits, and AcCYS1, an endogenous inhibitor of bromelain, in the development of blackheart. For this we checked the response to postharvest chilling treatment of two pineapple varieties (MD2 and Smooth Cayenne) differing in their resistance to blackheart. Quantitative RT-PCR analyses showed that postharvest chilling treatment induced a down-regulation of bromelain transcript accumulation in both varieties with the most dramatic drop in the resistant variety. Regarding AcCYS1 transcript accumulation, the varieties showed opposite trends with an up-regulation in the case of the resistant variety and a down-regulation in the susceptible one. Taken together our results suggest that the control of bromelain and AcCYS1 expression levels directly correlates to the resistance to blackheart development in pineapple fruits. Copyright © 2013 Elsevier GmbH. All rights reserved.

PineElm_SSRdb: a microsatellite marker database identified from genomic, chloroplast, mitochondrial and EST sequences of pineapple (Ananas comosus (L.) Merrill).

PubMed

Chaudhary, Sakshi; Mishra, Bharat Kumar; Vivek, Thiruvettai; Magadum, Santoshkumar; Yasin, Jeshima Khan

2016-01-01

Simple Sequence Repeats or microsatellites are resourceful molecular genetic markers. There are only few reports of SSR identification and development in pineapple. Complete genome sequence of pineapple available in the public domain can be used to develop numerous novel SSRs. Therefore, an attempt was made to identify SSRs from genomic, chloroplast, mitochondrial and EST sequences of pineapple which will help in deciphering genetic makeup of its germplasm resources. A total of 359511 SSRs were identified in pineapple (356385 from genome sequence, 45 from chloroplast sequence, 249 in mitochondrial sequence and 2832 from EST sequences). The list of EST-SSR markers and their details are available in the database. PineElm_SSRdb is an open source database available for non-commercial academic purpose at http://app.bioelm.com/ with a mapping tool which can develop circular maps of selected marker set. This database will be of immense use to breeders, researchers and graduates working on Ananas spp. and to others working on cross-species transferability of markers, investigating diversity, mapping and DNA fingerprinting.

Simulation and Failure Analysis of Car Bumper Made of Pineapple Leaf Fiber Reinforced Composite

NASA Astrophysics Data System (ADS)

Arbintarso, E. S.; Muslim, M.; Rusianto, T.

2018-02-01

The bumper car made of the Pineapple Leaf Fiber Reinforced Composite (PLFRC) is possible to be produced with the advantage of easy to get, and cheap. Pineapple leaf fiber has chosen as a natural fiber, which the maximum of the strength of 368 MPa. The objective of this study was to determine the maximum capability of front car bumpers using Pineapple Leaf Fiber Reinforced Composite materials through the process of simulating stress analysis with Solidworks 2014 software. The aim also to know the distribution of loads that occur on the front car bumper and predict the critical point position on the design of the bumper. The result will use to develop the alternative lightweight, cheap and environmentally friendly materials in general and the development of the use of pineapple fiber for automotive purposes in particular. Simulations and failure analysis have been conducted and showed an increased impact speed in line with increased displacement, strain, and stress that occur on the surface of the bumper. The bumper can withstand collisions at a speed of less than 70 kph.

Novel murine clonal cell lines either express slow or mixed (fast and slow) muscle markers following differentiation in vitro.

PubMed

Peltzer, J; Colman, L; Cebrian, J; Musa, H; Peckham, M; Keller, A

2008-05-01

We have investigated whether the phenotype of myogenic clones derived from satellite cells of different muscles from the transgenic immortomouse depended on muscle type origin. Clones derived from neonatal, or 6- to 12-week-old fast and slow muscles, were analyzed for myosin and enolase isoforms as phenotypic markers. All clones derived from slow-oxidative muscles differentiated into myotubes with a preferentially slow contractile phenotype, whereas some clones derived from rapid-glycolytic or neonatal muscles expressed both fast and slow myosin isoforms. Thus, muscle origin appears to bias myosin isoform expression in myotubes. The neonatal clone (WTt) was cultivated in various medium and substrate conditions, allowing us to determine optimized conditions for their differentiation. Matrigel allowed expressions of adult myosin isoforms, and an isozymic switch from embryonic alpha- toward muscle-specific beta-enolase, never previously observed in vitro. These cells will be a useful model for in vitro studies of muscle fiber maturation and plasticity.

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts.

PubMed

Brunetti, Dario; Perota, Andrea; Lagutina, Irina; Colleoni, Silvia; Duchi, Roberto; Calabrese, Fiorella; Seveso, Michela; Cozzi, Emanuele; Lazzari, Giovanna; Lucchini, Franco; Galli, Cesare

2008-12-01

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.

Benefits and problems with cloning animals.

PubMed Central

Smith, L C; Bordignon, V; Babkine, M; Fecteau, G; Keefer, C

2000-01-01

Animal cloning is becoming a useful technique for producing transgenic farm animals and is likely to be used to produce clones from valuable adults. Other applications will also undoubtedly be discovered in the near future, such as for preserving endangered breeds and species. Although cloning promises great advantages for commerce and research alike, its outcome is not always certain due to high pregnancy losses and high morbidity and mortality during the neonatal period. Research into the mechanisms involved in the reprogramming of the nucleus is being conducted throughout the world in an attempt to better understand the molecular and cellular mechanisms involved in correcting these problems. Although the cause of these anomalies remains mostly unknown, similar phenotypes have been observed in calves derived through in vitro fertilization, suggesting that culture conditions are involved in these phenomena. In the meantime, veterinarians and theriogenologists have an important role to play in improving the efficiency of cloning by finding treatments to assure normal gestation to term and to develop preventative and curative care for cloned neonates. Images Figure 1. PMID:11143925

Biotechnological Approaches in Plant Protection: Achievements, New Initiatives and Prospects

USDA-ARS?s Scientific Manuscript database

This chapter reviews the latest developments in the application of biotechnology to solve crop looses due to fungal disease. These include marker-assisted breeding, map-based cloning of genes, and development of transgenic plants. New areas of investigation which hold much promise are gene silenci...

Overexpression of wheat lipid transfer protein gene TaLTP5 increases resistances to Cochliobolus sativus and Fusarium graminearum in transgenic wheat.

PubMed

Zhu, Xiuliang; Li, Zhao; Xu, Huijun; Zhou, Miaoping; Du, Lipu; Zhang, Zengyan

2012-08-01

The fungus Cochliobolus sativus is the main pathogen of common root rot, a serious soil-borne disease of wheat (Triticum aestivum L.). The fungus Fusarium graminearum is the primary pathogen of Fusarium head blight, a devastating disease of wheat worldwide. In this study, the wheat lipid transfer protein gene, TaLTP5, was cloned and evaluated for its ability to suppress disease development in transgenic wheat. TaLTP5 expression was induced after C. sativus infection. The TaLTP5 expression vector, pA25-TaLTP5, was constructed and bombarded into Chinese wheat variety Yangmai 18. Six TaLTP5 transgenic wheat lines were established and characterized. PCR and Southern blot analyses indicated that the introduced TaLTP5 gene was integrated into the genomes of six transgenic wheat lines by distinct patterns, and heritable. RT-PCR and real-time quantitative RT-PCR revealed that the TaLTP5 gene was over-expressed in the transgenic wheat lines compared to segregants lacking the transgene and wild-type wheat plants. Following challenge with C. sativus or F. graminearum, all six transgenic lines overexpressing TaLTP5 exhibited significantly enhanced resistance to both common root rot and Fusarium head blight compared to the untransformed wheat Yangmai 18.

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins

PubMed Central

Kakitani, Makoto; Oshima, Takeshi; Horikoshi, Kaori; Yoshitome, Tetsuo; Ueda, Akiko; Kajikawa, Miwa; Iba, Yumi; Ozone, Yoshinao; Ijima, Yuki; Yoshino, Tohko; Itoh, Mikiko; Seki, Sachiko; Aoki, Ayako; Ishihara, Toshie; Shionoya, Michiyo; Makino, Utako; Kitada, Rina; Ohguma, Atsuko; Ohta, Takami; Yoshida, Yoshimasa; Kudoh, Hiroe; Hanaoka, Kazunori; Sibuya, Kazunori; Ishida, Isao; Kakeda, Minoru; Yagi, Mikio; Yoneya, Takashi; Tomizuka, Kazuma

2005-01-01

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) κ locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igκ region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras. PMID:15914664

Production of recombinant human lysozyme in the milk of transgenic pigs.

PubMed

Tong, Jia; Wei, HengXi; Liu, XiaoFang; Hu, WenPing; Bi, MingJun; Wang, YuanYuan; Li, QiuYan; Li, Ning

2011-04-01

In the swine industry pathogenic infections have a significant negative impact on neonatal survival. Piglets fed with human lysozyme, a natural antibiotic, might be more resistant to gastrointestinal infections. Here we describe the generation of transgenic swine expressing recombinant human lysozyme by somatic cell nuclear transfer. Three cloned female pigs were born, one of which expressed rhLZ at 0.32 ± 0.01 μg/ml in milk, 50-fold higher than that of the pig native lysozyme. Both the transgenic gilts and their progeny appear healthy. Introducing human lysozyme into pigs' milk has a potential to benefit the piglets by enhancing immune function and defending against pathogenic bacteria, thereby increasing the new born survival rate. This advance could be of great value to commercial swine producers.

Gluten-Free Recipes

MedlinePlus

... Yummy Easy Brownies Fruit Recipes Grilled Peaches Grilled Pineapple Recipe Grilled Watermelon Gluten Free Recipes "Fried" Onion ... Gravy for Thanksgiving Green Salad Grilled Peaches Grilled Pineapple Recipe Grilled Watermelon Gruyere Crustinis Haricots Verts Heavenly ...

Regulated expression of a calmodulin isoform alters growth and development in potato.

PubMed

Poovaiah, B W; Takezawa, D; An, G; Han, T J

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodulin isoform on plant growth and development. Eight genomic clones of potato calmodulin (PCM1 to 8) have been isolated and characterized (Takezawa et al., 1995). Among the potato calmodulin isoforms studied, PCM1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM1 fused to the CaMV 35S promoter. Transgenic plants showing a moderate increase in PCM1 mRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM1 mRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM1 mRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM1 protein in transgenic plants, indicating that the expression of both mRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM1 alters growth and development in potato plants.

High-Toughness Silk Produced by a Transgenic Silkworm Expressing Spider (Araneus ventricosus) Dragline Silk Protein

PubMed Central

Kuwana, Yoshihiko; Sezutsu, Hideki; Nakajima, Ken-ichi; Tamada, Yasushi; Kojima, Katsura

2014-01-01

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4–2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms. PMID:25162624

High-toughness silk produced by a transgenic silkworm expressing spider (Araneus ventricosus) dragline silk protein.

PubMed

Kuwana, Yoshihiko; Sezutsu, Hideki; Nakajima, Ken-ichi; Tamada, Yasushi; Kojima, Katsura

2014-01-01

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4-2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.

pHg/pSILBAγ vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA – triggering in the mycorrhizal fungus Laccaria bicolor

PubMed Central

Kemppainen, Minna J.; Pardo, Alejandro G.

2010-01-01

Summary pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy oriented two‐step PCR cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300‐based binary vector carrying a hygromycin resistance cassette, the pHg/pSILBAγ plasmid is used for Agrobacterium‐mediated transformation. The pHg/pSILBAγ system results in predominantly single integrations of RNA silencing triggering T‐DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. pSILBAγ construct and two other pSILBA plasmid variants (pSILBA and pSILBAα) were evaluated for their capacity to silence Laccaria nitrate reductase gene. While all pSILBA variants tested resulted in up to 65–76% of transformants with reduced growth on nitrate, pSILBAγ produced the highest number (65%) of strongly affected fungal strains. The strongly silenced phenotype was shown to correlate with T‐DNA integration in transcriptionally active genomic sites. pHg/pSILBAγ was shown to produce T‐DNAs with minimum CpG methylation in transgene promoter regions which assures the maximum silencing trigger production in Laccaria. Methylation of the target endogene was only slight in RNA silencing triggered with constructs carrying an intronic spacer hairpin sequence. The silencing capacity of the pHg/pSILBAγ was further tested with Laccaria inositol‐1,4,5‐triphosphate 5‐phosphatase gene. Besides its use in silencing triggering, the herein described plasmid system can also be used for transgene expression in Laccaria. pHg/pSILBAγ silencing system is optimized for L. bicolor but it should be highly useful also for other homobasidiomycetes, group of fungi currently lacking molecular tools for RNA silencing. PMID:21255319

Artificial cloning of domestic animals

PubMed Central

Keefer, Carol L.

2015-01-01

Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research. PMID:26195770

Artificial cloning of domestic animals.

PubMed

Keefer, Carol L

2015-07-21

Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.

RNAi-derived transgenic resistance to Mungbean yellow mosaic India virus in cowpea.

PubMed

Kumar, Sanjeev; Tanti, Bhaben; Patil, Basavaprabhu L; Mukherjee, Sunil Kumar; Sahoo, Lingaraj

2017-01-01

Cowpea is an important grain legume crop of Africa, Latin America, and Southeast Asia. Leaf curl and golden mosaic diseases caused by Mungbean yellow mosaic India virus (MYMIV) have emerged as most devastating viral diseases of cowpea in Southeast Asia. In this study, we employed RNA interference (RNAi) strategy to control cowpea-infecting MYMIV. For this, we generated transgenic cowpea plants harbouring three different intron hairpin RNAi constructs, containing the AC2, AC4 and fusion of AC2 and AC4 (AC2+AC4) of seven cowpea-infecting begomoviruses. The T0 and T1 transgenic cowpea lines of all the three constructs accumulated transgene-specific siRNAs. Transgenic plants were further assayed up to T1 generations, for resistance to MYMIV using agro-infectious clones. Nearly 100% resistance against MYMIV infection was observed in transgenic lines, expressing AC2-hp and AC2+AC4-hp RNA, when compared with untransformed controls and plants transformed with empty vectors, which developed severe viral disease symptoms within 3 weeks. The AC4-hp RNA expressing lines displayed appearance of milder symptoms after 5 weeks of MYMIV-inoculation. Northern blots revealed a positive correlation between the level of transgene-specific siRNAs accumulation and virus resistance. The MYMIV-resistant transgenic lines accumulated nearly zero or very low titres of viral DNA. The transgenic cowpea plants had normal phenotype with no yield penalty in greenhouse conditions. This is the first demonstration of RNAi-derived resistance to MYMIV in cowpea.

RNAi-derived transgenic resistance to Mungbean yellow mosaic India virus in cowpea

PubMed Central

Kumar, Sanjeev; Tanti, Bhaben; Patil, Basavaprabhu L.; Mukherjee, Sunil Kumar

2017-01-01

Cowpea is an important grain legume crop of Africa, Latin America, and Southeast Asia. Leaf curl and golden mosaic diseases caused by Mungbean yellow mosaic India virus (MYMIV) have emerged as most devastating viral diseases of cowpea in Southeast Asia. In this study, we employed RNA interference (RNAi) strategy to control cowpea-infecting MYMIV. For this, we generated transgenic cowpea plants harbouring three different intron hairpin RNAi constructs, containing the AC2, AC4 and fusion of AC2 and AC4 (AC2+AC4) of seven cowpea-infecting begomoviruses. The T0 and T1 transgenic cowpea lines of all the three constructs accumulated transgene-specific siRNAs. Transgenic plants were further assayed up to T1 generations, for resistance to MYMIV using agro-infectious clones. Nearly 100% resistance against MYMIV infection was observed in transgenic lines, expressing AC2-hp and AC2+AC4-hp RNA, when compared with untransformed controls and plants transformed with empty vectors, which developed severe viral disease symptoms within 3 weeks. The AC4-hp RNA expressing lines displayed appearance of milder symptoms after 5 weeks of MYMIV-inoculation. Northern blots revealed a positive correlation between the level of transgene-specific siRNAs accumulation and virus resistance. The MYMIV-resistant transgenic lines accumulated nearly zero or very low titres of viral DNA. The transgenic cowpea plants had normal phenotype with no yield penalty in greenhouse conditions. This is the first demonstration of RNAi-derived resistance to MYMIV in cowpea. PMID:29077738

Characterization of a Beta vulgaris PGIP defense gene promoter in transgenic plants

USDA-ARS?s Scientific Manuscript database

Polygalacturonase-inhibiting protein (BvPGIP) genes were cloned from a sugar beet breeding line F1016 with increased tolerance to the sugar beet root maggot. Polygalacturonase-inhibiting proteins are cell wall leucine-rich repeat (LRR) proteins with crucial roles in development, pathogen defense an...

Identical substitutions in magnesium chelatase paralogs result in chlorophyll deficient soybean mutants

USDA-ARS?s Scientific Manuscript database

The soybean (Glycine max (L.) Merr.) chlorophyll deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a non-synonymous nucleotide substitution in the third exon of a Mg-chelat...

Production of hydroxylated fatty acids in genetically modified plants

DOEpatents

Somerville, Chris; Broun, Pierre; van de Loo, Frank

2001-01-01

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants.

Applications of microencapsulated Bifidobacterium longum with Eleutherine americana in fresh milk tofu and pineapple juice.

PubMed

Phoem, Atchara N; Chanthachum, Suphitchaya; Voravuthikunchai, Supayang P

2015-04-03

Bifidobacterium longum was microencapsulated by extrusion technique and added in fresh milk tofu and pineapple juice. Microencapsulation of B. longum with Eleutherine americana extract, oligosaccharides extract, and commercial fructo-oligosaccharides was assessed for the bacterial survival after sequential exposure to simulated gastric and intestinal juices, and refrigeration storage. Microencapsulated B. longum with the extract and oligosaccharides extract in the food products showed better survival than free cells under adverse conditions. Sensory analysis demonstrated that the products containing co-encapsulated bacterial cells were more acceptable by consumers than free cells. Pineapple juice prepared with co-encapsulated cells had lower values for over acidification, compared with the juice with free cells added. This work suggested that microencapsulated B. longum with E. americana could enhance functional properties of fresh milk tofu and pineapple juice.

Applications of Microencapsulated Bifidobacterium Longum with Eleutherine Americana in Fresh Milk Tofu and Pineapple Juice

PubMed Central

Phoem, Atchara N.; Chanthachum, Suphitchaya; Voravuthikunchai, Supayang P.

2015-01-01

Bifidobacterium longum was microencapsulated by extrusion technique and added in fresh milk tofu and pineapple juice. Microencapsulation of B. longum with Eleutherine americana extract, oligosaccharides extract, and commercial fructo-oligosaccharides was assessed for the bacterial survival after sequential exposure to simulated gastric and intestinal juices, and refrigeration storage. Microencapsulated B. longum with the extract and oligosaccharides extract in the food products showed better survival than free cells under adverse conditions. Sensory analysis demonstrated that the products containing co-encapsulated bacterial cells were more acceptable by consumers than free cells. Pineapple juice prepared with co-encapsulated cells had lower values for over acidification, compared with the juice with free cells added. This work suggested that microencapsulated B. longum with E. americana could enhance functional properties of fresh milk tofu and pineapple juice. PMID:25854832

The addition of pineapple flesh and pineapple peels extracts to increase the quality of used cooking oil

NASA Astrophysics Data System (ADS)

Kusumawardani, R.; Hasanah, N.; Sukemi

2018-04-01

In Indonesia, reuse of cooking oil is high and common. Heating process and reuse of the cooking oil causes a change in its chemical constituents and decrease its qualities. This research aimed to investigate the addition of pineapple flesh extract (PFE) and pineapple peel extract (PPE) on the increment of the quality of oxidized (used) cooking oil. The cooking oil has been used three times. Treatment was done by mixing the used cooking oil with the extract (2:1) at 50°C. Peroxide value, FFA and iodine number of treated and untreated used cooking oils were measured by using titration method. The result showed that the treatment could increase the quality of the used cooking oils. PPE was better than PFE to increase the quality of the used cooking oil.

Polymer-based alternative method to extract bromelain from pineapple peel waste.

PubMed

Novaes, Letícia Celia de Lencastre; Ebinuma, Valéria de Carvalho Santos; Mazzola, Priscila Gava; Pessoa, Adalberto

2013-01-01

Bromelain is a mixture of proteolytic enzymes present in all tissues of the pineapple (Ananas comosus Merr.), and it is known for its clinical therapeutic applications, food processing, and as a dietary supplement. The use of pineapple waste for bromelain extraction is interesting from both an environmental and a commercial point of view, because the protease has relevant clinical potential. We aimed to study the optimization of bromelain extraction from pineapple waste, using the aqueous two-phase system formed by polyethylene glycol (PEG) and poly(acrylic acid). In this work, bromelain partitioned preferentially to the top/PEG-rich phase and, in the best condition, achieved a yield of 335.27% with a purification factor of 25.78. The statistical analysis showed that all variables analyzed were significant to the process. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Taboo in the Workplace: A Qualitative Study of Taiwanese Nurses' "Not-Eating Pineapple" in Clinical Practice.

PubMed

Tsai, Shu-Ling; Tsai, Chang-Hsiung; Hsu, Yu-Chien; Hsieh, Mei-Hui; Kao, Hsia-Tzu; Hsu, Min-Tao

There has been an increased emphasis on nurses' mental health and well-being in the workplace. Psychologists have established a correlative link between individual's beliefs on luck and mental health. The pineapple taboo has been observed among Taiwanese hospital nurses as a prevalent superstitious belief for bringing luck or warding off increased clinical workloads, but how and why the ritual persists in the hospital workplace remains unknown. This article aims to explore the latent meaning of observance of the taboo and how it is related to nurses' clinical practice and possibly affects their mental health at work. A qualitative research was designed in line with the hermeneutic phenomenological method. Through purposive sampling, 18 nurse participants were recruited for in-depth semistructured interviews. Resulting from the ensuing analysis, 3 modalities were identified as constituting the spectrum of observance of the taboo: (a) "strictly not eating pineapple"; (b) "not eating pineapple at work"; and (c) "eating pineapple without admitting to doing so." Each reflects the position of nurses revealed in relation to the pineapple taboo in clinical settings. Based on the subjective narratives of nurses, it may be understood as an active moral attempt at "being right" rather than a passive avoidance of bad luck in the taboo observation. The findings facilitate an appropriate understanding of the embedded meaning of nurses' workplace-related belief and its seminal function of empowerment for nurses in holistic nursing practice.

In vitro Study of Noni Juice Extract Waste (Morinda citrifolia L.) and Pineapple Industrial Wastes (Ananas comosus L. Merr) as Energy Supplement in Dairy Goat Ration

NASA Astrophysics Data System (ADS)

Evvyernie, D.; Tjakradidjaja, A. S.; Permana, I. G.; Toharmat, T.; Insani, A.

2018-02-01

The aim of the study was to evaluate the potency of noni juice extract waste (Morinda citrifolia L.) and pineapple industrial wastes (Ananas comosus L. Merr) as an energy supplement in dairy goat ration through in vitro study. This study used a complete randomized design with 5 treatments and 3 rumen fluid groups. The treatments were R0 as control (60% Napier grass (NG) + 40% concentrate), R1 (45% NG + 15% noni juice extract waste + 40% concentrate) + R2 (45% NG + 15% noni juice extract waste ammoniated + 40% concentrate), R3 (45% NG + 15% pineapple peel + 40% concentrate), and R4 (45% NG + 15% pineapple crown + 40% concentrate). The variables were totalbacterial population, protozoal population, fermentation characteristic (total VFA and NH3 concentration), and digestibility (dry matter and organic matter).Data were analyzed with analysis of variance (ANOVA) and differences among treatments were determined by orthogonal contrast.The results showed that total VFA concentration was significant increased (P<0.05) by replacing 25% napier grass with noni juice extract waste (R1)or very significant increased(P<0.01) by replacing the grass with pineapple peel (R3). The average increasing of total VFA concentration was 74% compared to control. As conclusions, 15% pineapple peel or 15% noni juice extract waste can use as an energy supplement by replacing 25% of napier grass in lactating dairy goat ration.

Positive and Negative Selection in Transgenic Mice Expressing a T-Cell Receptor Specific for Influenza Nucleoprotein and Endogenous Superantigen

PubMed Central

Mamalaki, Clio; Elliott, James; Norton, Trisha; Yannoutsos, Nicholas; Townsend, Alain R.; Chandler, Phillip; Simpson, Elizabeth

1993-01-01

A transgenic mouse was generated expressing on most (>80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8+ and recognizes αα366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class ,MHC Db (Townsend et al., 1986). The receptor utilizes the Vβ11 and Vα4 gene segments for the β chain and α chain, respectively (Palmer et al., 1989). The usage of Vβ11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2b, IE-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2k and BALB/c H-2d) and the endogenous Mtv ligands. Positive selection of CD8+ T cells expressing the Vβ11 is seen in C57BL/10 transgenic mice (H-2b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period of in vitro culture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8+ peripheral T-cells in these (H-2k or H-2d) F5 mice are predominantly Vβ11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2d scid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired. PMID:8281031

Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

PubMed

Lardizabal, K D; Metz, J G; Sakamoto, T; Hutton, W C; Pollard, M R; Lassner, M W

2000-03-01

Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

PubMed

Patade, Vikas Yadav; Khatri, Deepti; Kumar, Kamal; Grover, Atul; Kumari, Maya; Gupta, Sanjay Mohan; Kumar, Devender; Nasim, Mohammed

2014-07-01

Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

Development of disease-resistant marker-free tomato by R/RS site-specific recombination.

PubMed

Khan, Raham Sher; Nakamura, Ikuo; Mii, Masahiro

2011-06-01

The selection marker genes, imparting antibiotic or herbicide resistance, in the final transgenics have been criticized by the public and considered a hindrance in their commercialization. Multi-auto-transformation (MAT) vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators (PGRs). In the study reported here, isopentenyltransferase (ipt) gene was used as a selection marker and wasabi defensin (WD) gene, isolated from Wasabia japonica as a target gene. WD was cloned from the binary vector, pEKH-WD to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledons of tomato cv. Reiyo were cultured on PGR- and antibiotic-free MS medium. Adventitious shoots were developed by the explants infected with the pMAT21/wasabi defensin. The same PGR- and antibiotic-free MS medium was used in subcultures of the adventitious shoot lines (ASLs) to produce ipt and normal shoots. Approximately, 6 months after infection morphologically normal shoots were produced. Molecular analyses of the developed shoots confirmed the integration of gene of interest (WD) and excision of the selection marker (ipt). Expression of WD was confirmed by Northern blot and Western blot analyses. The marker-free transgenic plants exhibited enhanced resistance against Botrytis cinerea (gray mold), Alternaria solani (early blight), Fusarium oxysporum (Fusarium wilt) and Erysiphe lycopersici (powdery mildew).

Influence of harvest maturity and fruit logistics on pineapple (Ananas comosus [L.] Merr.) volatiles assessed by headspace solid phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC/MS).

PubMed

Steingass, Christof B; Grauwet, Tara; Carle, Reinhold

2014-05-01

Profiling of volatiles from pineapple fruits was performed at four ripening stages using headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC/MS). In total, 142 volatiles were detected, of which 132 were identified. Multivariate data analysis was carried out to assess the effect of post-harvest storage on volatiles composition of green-ripe sea-freighted pineapple in comparison to air-freighted fruits harvested at full maturity. The latter fruits were characterised by volatiles described as potent odorants in pineapples, such as δ-octalactone, γ-lactones, 1-(E,Z)-3,5-undecatriene and 1,3,5,8-undecatetraene, as well as various methyl esters. In contrast, post-harvest storage of green-ripe sea-freighted fruits resulted in an increased formation of ethyl esters, acetates, acetoxy esters and alcohols, thus allowing the authentication of sea- and air-freighted pineapples, respectively. Particularly, compounds presumably derived from methyl-branched amino acid catabolism were identified in the fruits at later post-harvest stages. In addition, physicochemical traits were determined to characterise the fruit maturity stages. Copyright © 2013 Elsevier Ltd. All rights reserved.

DNA profiling of pineapple cultivars in Japan discriminated by SSR markers

PubMed Central

Shoda, Moriyuki; Urasaki, Naoya; Sakiyama, Sumisu; Terakami, Shingo; Hosaka, Fumiko; Shigeta, Narumi; Nishitani, Chikako; Yamamoto, Toshiya

2012-01-01

We developed 18 polymorphic simple sequence repeat (SSR) markers in pineapple (Ananas comosus) by using genomic libraries enriched for GA and CA motifs. The markers were used to genotype 31 pineapple accessions, including seven cultivars and 11 breeding lines from Okinawa Prefecture, 12 foreign accessions and one from a related species. These SSR loci were highly polymorphic: the 31 accessions contained three to seven alleles per locus, with an average of 4.1. The values of expected heterozygosity ranged from 0.09 to 0.76, with an average of 0.52. All 31 accessions could be successfully differentiated by the 18 SSR markers, with the exception of ‘N67-10’ and ‘Hawaiian Smooth Cayenne’. A single combination of three markers TsuAC004, TsuAC010 and TsuAC041, was enough to distinguish all accessions with one exception. A phenogram based on the SSR genotypes did not show any distinct groups, but it suggested that pineapples bred in Japan are genetically diversed. We reconfirmed the parentage of 14 pineapple accessions by comparing the SSR alleles at 17 SSR loci in each accession and its reported parents. The obtained information will contribute substantially to protecting plant breeders’ rights. PMID:23341750

Authentication of pineapple (Ananas comosus [L.] Merr.) fruit maturity stages by quantitative analysis of γ- and δ-lactones using headspace solid-phase microextraction and chirospecific gas chromatography-selected ion monitoring mass spectrometry (HS-SPME-GC-SIM-MS).

PubMed

Steingass, Christof B; Langen, Johannes; Carle, Reinhold; Schmarr, Hans-Georg

2015-02-01

Headspace solid phase microextraction and chirospecific gas chromatography-mass spectrometry in selected ion monitoring mode (HS-SPME-GC-SIM-MS) allowed quantitative determination of δ-lactones (δ-C8, δ-C10) and γ-lactones (γ-C6, γ-C8, γ-C10). A stable isotope dilution assay (SIDA) with d7-γ-decalactone as internal standard was used for quantitative analysis of pineapple lactones that was performed at three progressing post-harvest stages of fully ripe air-freighted and green-ripe sea-freighted fruits, covering the relevant shelf-life of the fruits. Fresh pineapples harvested at full maturity were characterised by γ-C6 of high enantiomeric purity remaining stable during the whole post-harvest period. In contrast, the enantiomeric purity of γ-C6 significantly decreased during post-harvest storage of sea-freighted pineapples. The biogenetical background and the potential of chirospecific analysis of lactones for authentication and quality evaluation of fresh pineapple fruits are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

Physico-chemical properties, antioxidant activity and mineral contents of pineapple genotypes grown in china.

PubMed

Lu, Xin-Hua; Sun, De-Quan; Wu, Qing-Song; Liu, Sheng-Hui; Sun, Guang-Ming

2014-06-23

The fruit physico-chemical properties, antioxidant activity and mineral contents of 26 pineapple [Ananas comosus (L.) Merr.] genotypes grown in China were measured. The results showed great quantitative differences in the composition of these pineapple genotypes. Sucrose was the dominant sugar in all 26 genotypes, while citric acid was the principal organic acid. Potassium, calcium and magnesium were the major mineral constituents. The ascorbic acid (AsA) content ranged from 5.08 to 33.57 mg/100 g fresh weight (FW), while the total phenolic (TP) content varied from 31.48 to 77.55 mg gallic acid equivalents (GAE)/100 g FW. The two parameters in the predominant cultivars Comte de Paris and Smooth Cayenne were relative low. However, MD-2 indicated the highest AsA and TP contents (33.57 mg/100 g and 77.55 mg GAE/100 g FM, respectively), and it also showed the strongest antioxidant capacity 22.85 and 17.30 μmol TE/g FW using DPPH and TEAC methods, respectively. The antioxidant capacity of pineapple was correlated with the contents of phenolics, flavonoids and AsA. The present study provided important information for the further application of those pineapple genotypes.

Pineapple by-product and canola oil as partial fat replacers in low-fat beef burger: Effects on oxidative stability, cholesterol content and fatty acid profile.

PubMed

Selani, Miriam M; Shirado, Giovanna A N; Margiotta, Gregório B; Rasera, Mariana L; Marabesi, Amanda C; Piedade, Sonia M S; Contreras-Castillo, Carmen J; Canniatti-Brazaca, Solange G

2016-05-01

The effect of freeze-dried pineapple by-product and canola oil as fat replacers on the oxidative stability, cholesterol content and fatty acid profile of low-fat beef burgers was evaluated. Five treatments were performed: conventional (CN, 20% fat) and four low-fat formulations (10% fat): control (CT), pineapple by-product (PA), canola oil (CO), and pineapple by-product and canola oil (PC). Low-fat cooked burgers showed a mean cholesterol content reduction of 9.15% compared to the CN. Canola oil addition improved the fatty acid profile of the burgers, with increase in the polyunsaturated/saturated fatty acids ratio and decrease in the n-6/n-3 ratio, in the atherogenic and thrombogenic indexes. The oxidative stability of the burgers was affected by the vegetable oil addition. However, at the end of the storage time (120 days), malonaldehyde values of CO and PC were lower than the threshold for the consumer's acceptance. Canola oil, in combination with pineapple by-product, can be considered promising fat replacers in the development of healthier burgers. Copyright © 2016 Elsevier Ltd. All rights reserved.

Physical and chemical pretreatment of lignocellulosics in pineapple (Ananus comosus) peels dried for investment

NASA Astrophysics Data System (ADS)

Sukkaew, Adulsman; Boonsong, Panthip; Thongpradistha, Sriubol; Intan, Maimoon

2017-08-01

Pineapple (Ananus comosus) Peels, once known as waste from agricultural, can be a problem when we eliminate in agriculture and industry. The current technology can help preliminarily to solve this problem. The sustainable solution to this problem is lignocellulosics pretreatments for converted saccharide as a carbon source for ethanol production. The objective of this study is the investigation of pineapple peels pretreatment to produce fermentable sugar by drying and digesting 5% sulfuric acid (H2SO4). And study of cost economic passed selection for investment. The result found that the best investment of drying was 100 °C at 11 hours for the sulphuric acid which could be easily crushed into a fine powder. Moreover, digestion of pineapple peels gave the best total sugar 252.2 g/l by 5% H2SO4 incubated for 60 minutes at room temperature. The pineapple peels were digested by 5%H2SO4 concentration by incubating for 60 minutes at room temperature, finding to be the best condition and the lowest investment. Finally, the optimisation of investment and management for lignocellulosic pretreatment will improve efficiency of strategy for economic and energy development.

Cloning and Functional Analysis of Phosphoethanolamine Methyltransferase Promoter from Maize (Zea mays L.)

PubMed Central

Niu, Gai-Li; Gou, Wei; Han, Xiang-Long; Qin, Cheng; Zhang, Li-Xin; Ashraf, Muhammad

2018-01-01

Betaine, a non-toxic osmoprotectant, is believed to accumulate considerably in plants under stress conditions to maintain the osmotic pressure and promote a variety of processes involved in growth and development. Phosphoethanolamine N-methyltransferase (PEAMT), a key enzyme for betaine synthesis, is reported to be regulated by its upstream promoter. In the present investigation, by using the transgenic approach, a 1048 bp long promoter region of ZmPEAMT gene from Zea mays was cloned and functionally characterized in tobacco. Computational analysis affirmed the existence of abiotic stress responsive cis-elements like ABRE, MYC, HST, LST etc., as well as pathogen, wound and phytohormone responsive motifs. For transformation in tobacco, four 5′-deletion constructs of 826 bp (P2), 642 bp (P3), 428 bp (P4) and 245 bp (P5) were constructed from the 1048 bp (P1) promoter fragment. The transgenic plants generated through a single event exhibited a promising expression of GUS reporter protein in the leaf tissues of treated with salt, drought, oxidative and cold stress as well as control plants. The GUS expression level progressively reduced from P1 to P5 in the leaf tissues, whereas a maximal expression was observed with the P3 construct in the leaves of control plants. The expression of GUS was noted to be higher in the leaves of osmotically- or salt-treated transgenic plants than that in the untreated (control) plants. An effective expression of GUS in the transgenic plants manifests that this promoter can be employed for both stress-inducible and constitutive expression of gene(s). Due to this characteristic, this potential promoter can be effectively used for genetic engineering of several crops. PMID:29316727

Generation of α-1,3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes.

PubMed

Kwon, Dae-Jin; Kim, Dong-Hwan; Hwang, In-Sul; Kim, Dong-Ern; Kim, Hyung-Joo; Kim, Jang-Seong; Lee, Kichoon; Im, Gi-Sun; Lee, Jeong-Woong; Hwang, Seongsoo

2017-02-01

Recent progress in genetic manipulation of pigs designated for xenotransplantation ha6s shown considerable promise on xenograft survival in primates. However, genetic modification of multiple genes in donor pigs by knock-out and knock-in technologies, aiming to enhance immunological tolerance against transplanted organs in the recipients, has not been evaluated for health issues of donor pigs. We produced transgenic Massachusetts General Hospital piglets by knocking-out the α-1,3-galactosyltransferase (GT) gene and by simultaneously knocking-in an expression cassette containing five different human genes including, DAF, CD39, TFPI, C1 inhibitor (C1-INH), and TNFAIP3 (A20) [GT -(DAF/CD39/TFPI/C1-INH/TNFAIP3)/+ ] that are connected by 2A peptide cleavage sequences to release individual proteins from a single translational product. All five individual protein products were successfully produced as determined by western blotting of umbilical cords from the newborn transgenic pigs. Although gross observation and histological examination revealed no significant pathological abnormality in transgenic piglets, hematological examination found that the transgenic piglets had abnormally low numbers of platelets and WBCs, including neutrophils, eosinophils, basophils, and lymphocytes. However, transgenic piglets had similar numbers of RBC and values of parameters related to RBC compared to the control littermate piglets. These data suggest that transgenic expression of those human genes in pigs impaired hematopoiesis except for erythropoiesis. In conclusion, our data suggest that transgenic expression of up to five different genes can be efficiently achieved and provide the basis for determining optimal dosages of transgene expression and combinations of the transgenes to warrant production of transgenic donor pigs without health issues.

Development of the gateway recycling cloning system for multiple linking of expression cassettes in a defined order, and direction on gateway compatible binary vectors.

PubMed

Kimura, Tetsuya; Nakao, Akihide; Murata, Sachiko; Kobayashi, Yasuyuki; Tanaka, Yuji; Shibahara, Kenta; Kawazu, Tetsu; Nakagawa, Tsuyoshi

2013-01-01

We developed the Gateway recycling cloning system, which allows multiple linking of expression cassettes by multiple rounds of the Gateway LR reaction. Employing this system, the recycling donor vector pRED419 was subjected to the first LR reaction with an attR1-attR2 type destination vector. Then conversion vector pCON was subjected to an LR reaction to restore the attR1-attR2 site on the destination vector for the next cloning cycle. By repetition of these two simple steps, we linked four expression cassettes of a reporter gene in Gateway binary vector pGWB1, introduced the constructs into tobacco BY-2 cells, and observed the expression of transgenes.

Survival of SA11 rotavirus in fresh fruit juices of pineapple, papaya, and honeydew melon.

PubMed

Leong, Yap Kok; Xui, Ong Chiaw; Chia, Ong Kien

2008-05-01

Survival of rotavirus in fresh fruit juices of papaya (Caraca papaya L.), honeydew melon (Cucumis melo L.), and pineapple (Ananas comosus [L.] Merr.) was studied. Clarified juices were prepared from pulps of ripe fruits and sterilized by ultrafiltration. One milliliter of juice from each fruit was inoculated with 20 microl of 1 x 10(6) PFU of SA11 rotavirus and sampled immediately (0-h exposure) and 1 and 3 h later at 28 degrees C. Mean viral titers in juices of papaya (pH 5.1) and honeydew melon (pH 6.3) at 1 and 3 h were not significantly different from titers at 0-h exposure. Mean viral titers in juices from pineapples with ripening color indices of 3 (pH 3.6) and 6 (pH 3.7) at 1-h exposure (color index 3: 4.0 +/- 1.7 x 10(4); color index 6: 2.3 +/- 0.3 x 10(5)) and 3-h exposure (color index 3: 1.1 +/- 0.4 x 10(4); color index 6:1.3 +/- 0.6 x 10(5)) were significantly lower than titers at 0-h exposure (color index 3: 5.7 +/- 2.9 x 10(5); color index 6: 7.4 +/- 1.3 x 10(5)). Virus titers in pineapple juices of color index 3 were significantly lower than titers of the virus in juices of index 6. In cell culture medium (pH 7.4), SA11 titer remained stable over 3 h at 28 degrees C. However, at pH 3.6, the virus titer was reduced to a level not significantly different from that of the virus in pineapple juice of color index 6 (pH 3.7). In conclusion, papaya and honeydew melon juices, in contrast to pineapple juice, have the potential to transmit rotavirus. Inactivation of SA11 virus in pineapple juice can be possibly attributed to low pH and constituent(s) in the juice.

Coupled social and ecological outcomes of land use change and agricultural intensification in Costa Rica and the future of biodiversity conservation in tropical agricultural regions.

NASA Astrophysics Data System (ADS)

Sanfiorenzo, A. R.; Shaver, I.; Chain Guadarrama, A.; Cleary, K.; Santiago-Garcia, R.; Finegan, B.; Hormel, L.; Sibelet, N.; Vierling, L. A.; Bosque-Perez, N.; DeClerck, F.; Fagan, M. E.; Waits, L.

2016-12-01

Tropical ecosystem conversion to agriculture has caused widespread habitat loss and created fragmented landscapes composed of remnant forest patches embedded in a matrix of agricultural land uses. Non- traditional agricultural export (NTAE) crops such as pineapple are rapidly replacing multiuse landscapes characterized by a diverse matrix of pasture and smallholder crops with intensive, large-scale, monoculture plantations. Using an interdisciplinary approach, we conduct a case study to examine the coupled social and ecological implications of LUCC and agricultural intensification in this region, with larger application to regions experiencing similar patterns. Guided by frameworks from both political and landscape ecology, we: (1) describe the social and economic implications of pineapple expansion, specifically the concentration of land, labor and financial resources, (2) quantify pineapple cultivation's spatial characteristics, and (3) assess the effects of pineapple expansion on surrounding forest ecosystems, on the agricultural matrix and on biodiversity conservation. Our results indicate that pineapple production concentrates land, labor, and financial resources, which has a homogenizing effect on the agricultural economy in the study region. This constrains farm-based livelihoods, with larger implications for food security and agricultural diversity. Landscape ecology analyses further reveal how pineapple production simplifies and homogenizes the agricultural matrix between forest patches, which is likely to have a negative effect on biodiversity. To offset the effects of pineapple expansion on social and environmental systems, we recommend developing landscape level land use planning capacity. Furthermore, agricultural and conservation policy reform is needed to promote landscape heterogeneity and economic diversity within the agricultural sector. Our interdisciplinary research provides a detailed examination of the social and ecological impacts of agricultural intensification in a tropical landscape, and offers recommendations for improvement relevant not only to our study region but to the many other tropical landscapes currently undergoing non-traditional agricultural export driven agricultural intensification.

Coupled social and ecological outcomes of land use change and agricultural intensification in Costa Rica and the future of biodiversity conservation in tropical agricultural regions.

NASA Astrophysics Data System (ADS)

Sanfiorenzo, A. R.; Shaver, I.; Chain Guadarrama, A.; Cleary, K.; Santiago-Garcia, R.; Finegan, B.; Hormel, L.; Sibelet, N.; Vierling, L. A.; Bosque-Perez, N.; DeClerck, F.; Fagan, M. E.; Waits, L.

2017-12-01

Tropical ecosystem conversion to agriculture has caused widespread habitat loss and created fragmented landscapes composed of remnant forest patches embedded in a matrix of agricultural land uses. Non- traditional agricultural export (NTAE) crops such as pineapple are rapidly replacing multiuse landscapes characterized by a diverse matrix of pasture and smallholder crops with intensive, large-scale, monoculture plantations. Using an interdisciplinary approach, we conduct a case study to examine the coupled social and ecological implications of LUCC and agricultural intensification in this region, with larger application to regions experiencing similar patterns. Guided by frameworks from both political and landscape ecology, we: (1) describe the social and economic implications of pineapple expansion, specifically the concentration of land, labor and financial resources, (2) quantify pineapple cultivation's spatial characteristics, and (3) assess the effects of pineapple expansion on surrounding forest ecosystems, on the agricultural matrix and on biodiversity conservation. Our results indicate that pineapple production concentrates land, labor, and financial resources, which has a homogenizing effect on the agricultural economy in the study region. This constrains farm-based livelihoods, with larger implications for food security and agricultural diversity. Landscape ecology analyses further reveal how pineapple production simplifies and homogenizes the agricultural matrix between forest patches, which is likely to have a negative effect on biodiversity. To offset the effects of pineapple expansion on social and environmental systems, we recommend developing landscape level land use planning capacity. Furthermore, agricultural and conservation policy reform is needed to promote landscape heterogeneity and economic diversity within the agricultural sector. Our interdisciplinary research provides a detailed examination of the social and ecological impacts of agricultural intensification in a tropical landscape, and offers recommendations for improvement relevant not only to our study region but to the many other tropical landscapes currently undergoing non-traditional agricultural export driven agricultural intensification.

Ancestral genomic duplication of the insulin gene in tilapia: An analysis of possible implications for clinical islet xenotransplantation using donor islets from transgenic tilapia expressing a humanized insulin gene.

PubMed

Hrytsenko, Olga; Pohajdak, Bill; Wright, James R

2016-07-03

Tilapia, a teleost fish, have multiple large anatomically discrete islets which are easy to harvest, and when transplanted into diabetic murine recipients, provide normoglycemia and mammalian-like glucose tolerance profiles. Tilapia insulin differs structurally from human insulin which could preclude their use as islet donors for xenotransplantation. Therefore, we produced transgenic tilapia with islets expressing a humanized insulin gene. It is now known that fish genomes may possess an ancestral duplication and so tilapia may have a second insulin gene. Therefore, we cloned, sequenced, and characterized the tilapia insulin 2 transcript and found that its expression is negligible in islets, is not islet-specific, and would not likely need to be silenced in our transgenic fish.

Ancestral genomic duplication of the insulin gene in tilapia: An analysis of possible implications for clinical islet xenotransplantation using donor islets from transgenic tilapia expressing a humanized insulin gene

PubMed Central

Hrytsenko, Olga; Pohajdak, Bill; Wright, James R.

2016-01-01

ABSTRACT Tilapia, a teleost fish, have multiple large anatomically discrete islets which are easy to harvest, and when transplanted into diabetic murine recipients, provide normoglycemia and mammalian-like glucose tolerance profiles. Tilapia insulin differs structurally from human insulin which could preclude their use as islet donors for xenotransplantation. Therefore, we produced transgenic tilapia with islets expressing a humanized insulin gene. It is now known that fish genomes may possess an ancestral duplication and so tilapia may have a second insulin gene. Therefore, we cloned, sequenced, and characterized the tilapia insulin 2 transcript and found that its expression is negligible in islets, is not islet-specific, and would not likely need to be silenced in our transgenic fish. PMID:27222321

Group Cohesiveness, Deviation, Stress, and Conformity

DTIC Science & Technology

1993-08-11

Cola number of cups, __ Chocolate, cocoa, wine, beer/alcohol, decaffeinated coffee. Breads containing raisins, prunes, orange peel , banana , or...pudding, mince pie. Banana , avocado, pineapple, canned figs, raisins, plums and prunes. Oranges, orange juice, fru~ cocktail with pineapple. Tomato

Physicochemical changes of 'Phulae' pineapple fruit treated with short-term anoxia during ambient storage.

PubMed

Techavuthiporn, Chairat; Boonyaritthongchai, Panida; Supabvanich, Suriyan

2017-08-01

The effects of short-term anoxia exposure for 16h on physicochemical changes of 'Phulae' pineapple fruit stored at ambient temperature (25±2°C) were investigated. The respiratory rate of the fruit was induced by the anoxia treatment. However, it retarded the increase in moisture loss and maintained both flesh and pulp colour by inhibiting polyphenol oxidase (PPO) activity of the both tissues. The anoxia exposure delayed the increase in total sugar content and enhanced total ascorbic acid content during storage. The half-cut pineapple fruit showed that the anoxia exposure completely inhibited internal transparency of the flesh tissue adjacent to core during the storage. In conclusion, the short-term anoxia exposure for 16h maintained postharvest quality, retarded physiological disorder and enhanced nutritional values of the pineapple fruit stored at ambient temperature (25±2°C). Copyright © 2017 Elsevier Ltd. All rights reserved.

Pineapple juice and its fractions in enzymatic browning inhibition of banana [Musa (AAA group) Gros Michel].

PubMed

Chaisakdanugull, Chitsuda; Theerakulkait, Chockchai; Wrolstad, Ronald E

2007-05-16

The effectiveness of pineapple juice in enzymatic browning inhibition was evaluated on the cut surface of banana slices. After storage of banana slices at 15 degrees C for 3 days, pineapple juice showed browning inhibition to a similar extent as 8 mM ascorbic acid but less than 4 mM sodium metabisulfite. Fractionation of pineapple juice by a solid-phase C18 cartridge revealed that the directly eluted fraction (DE fraction) inhibited banana polyphenol oxidase (PPO) about 100% when compared to the control. The DE fraction also showed more inhibitory effect than 8 mM ascorbic acid in enzymatic browning inhibition of banana puree during storage at 5 degrees C for 24 h. Further identification of the DE fraction by fractionation with ion exchange chromatography and confirmation using model systems indicated that malic acid and citric acid play an important role in the enzymatic browning inhibition of banana PPO.

Generation and phenotypic analysis of a transgenic line of rabbits secreting active recombinant human erythropoietin in the milk.

PubMed

Mikus, Tomás; Poplstein, Martin; Sedláková, Jirina; Landa, Vladimír; Jeníkova, Gabriela; Trefil, Pavel; Lidický, Jan; Malý, Petr

2004-10-01

Production of recombinant human erythropoietin (rhEPO) for therapeutic purposes relies on its expression in selected clones of transfected mammalian cells. Alternatively, this glycoprotein can be produced by targeted secretion into the body fluid of transgenic mammals. Here, we report on the generation of a transgenic rabbits producing rhEPO in the lactating mammary gland. Transgenic individuals are viable, fertile and transmit the rhEPO gene to the offspring. Northern blot data indicated that the expression of the transgene in the mammary gland is controlled by whey acidic protien (WAP) regulatory sequences during the period of lactation. While the hybridization with total RNA revealed the expression only in the lactating mammary gland, the highly sensitive combinatory approach using RT-PCR/hybridization technique detected a minor ectopic expression. The level of rhEPO secretion in the founder female, measured in the period of lactation, varied in the range of 60-178 and 60-162 mIU/ml in the milk and blood plasma, respectively. Biological activity of the milk rhEPO was confirmed by a standard [3H]-thymidine incorporation test. Thus, we describe the model of a rhEPO-transgenic rabbit, valuable for studies of rhEPO glycosylation and function, which can be useful for the development of transgenic approaches designed for the preparation of recombinant proteins by alternative biopharmaceutical production.

Expression of hemagglutinin protein of Rinderpest virus in transgenic pigeon pea [Cajanus cajan (L.) Millsp.] plants.

PubMed

Satyavathi, V V; Prasad, V; Khandelwal, Abha; Shaila, M S; Sita, G Lakshmi

2003-03-01

Rinderpest virus is the causative agent of a devastating, often fatal disease in wild and domestic bovids that is endemic in Africa, the Middle East and South Asia. The existing live attenuated vaccine is heat-labile, and thus there is a need for the development of new strategies for vaccination. This paper reports the development of transgenic pigeon pea [ Cajanus cajun (L.) Millsp.] expressing one of the protective antigens, the hemagglutinin (H) protein of Rinderpest virus. A 2-kb fragment containing the coding region of the H protein was cloned into pBI121 and mobilized into Agrobacterium tumefaciensstrain EHA105. Embryonic axes and cotyledonary nodes from germinated seeds of pigeon pea were used for transformation. The presence of the transgene in transgenic plants was confirmed by Southern blots, and the specific transcription of the marker gene in the plants was demonstrated by reverse transcription-polymerase chain reaction. Integration of the H gene into the pigeon pea genome was confirmed by Southern hybridization. The expression of the H protein in the transgenic lines was confirmed by Western blot analysis using a polyclonal monospecific antibody to the H protein. The highest level of expression of the hemagglutinin protein in leaves of pigeon pea was 0.49% of the total soluble protein. The transgenic plants were fertile and the transgene expressed in the progeny.

Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

PubMed Central

Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

1994-01-01

We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

Improved forage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase.

PubMed

Chen, Lei; Auh, Chung-Kyoon; Dowling, Paul; Bell, Jeremey; Chen, Fang; Hopkins, Andrew; Dixon, Richard A; Wang, Zeng-Yu

2003-11-01

Lignification of cell walls during plant development has been identified as the major factor limiting forage digestibility and concomitantly animal productivity. cDNA sequences encoding a key lignin biosynthetic enzyme, cinnamyl alcohol dehydrogenase (CAD), were cloned from the widely grown monocotyledonous forage species tall fescue (Festuca arundinacea Schreb.). Recombinant tall fescue CAD expressed in E. coli exhibited the highest V(max)/K(m) values when coniferaldehyde and sinapaldehyde were used as substrates. Transgenic tall fescue plants carrying either sense or antisense CAD gene constructs were obtained by microprojectile bombardment of single genotype-derived embryogenic suspension cells. Severely reduced levels of mRNA transcripts and significantly reduced CAD enzymatic activities were found in two transgenic plants carrying sense and antisense CAD transgenes, respectively. These CAD down-regulated transgenic lines had significantly decreased lignin content and altered ratios of syringyl (S) to guaiacyl (G), G to p-hydroxyphenyl (H) and S to H units. No significant changes in cellulose, hemicellulose, neutral sugar composition, p-coumaric acid and ferulic acid levels were observed in the transgenic plants. Increases of in vitro dry matter digestibility of 7.2-9.5% were achieved in the CAD down-regulated lines, thus providing a novel germplasm to be used for the development of grass cultivars with improved forage quality.

Postprandial glucose response to selected tropical fruits in normal glucose-tolerant Nigerians.

PubMed

Edo, A; Eregie, A; Adediran, O; Ohwovoriole, A; Ebengho, S

2011-01-01

The glycemic response to commonly eaten fruits in Nigeria has not been reported. Therefore, this study assessed the plasma glucose response to selected fruits in Nigeria. Ten normal glucose-tolerant subjects randomly consumed 50 g carbohydrate portions of three fruits: banana (Musa paradisiaca), pineapple (Ananus comosus), and pawpaw (Carica papaya), and a 50-g glucose load at 1-week intervals. Blood samples were collected in the fasting state and half-hourly over a 2-h period post-ingestion of the fruits or glucose. The samples were analyzed for plasma glucose concentrations. Plasma glucose responses were assessed by the peak plasma glucose concentration, maximum increase in plasma glucose, 2-h postprandial plasma glucose level, and incremental area under the glucose curve and glycemic index (GI). The results showed that the blood glucose response to these three fruits was similar in terms of their incremental areas under the glucose curve, maximum increase in plasma glucose, and glycemic indices (GIs). The 2-h postprandial plasma glucose level of banana was significantly higher than that of pineapple, P < 0.025. The mean ± SEM GI values were as follows: pawpaw; 86 ± 26.8%; banana, 75.1 ± 21.8%; pineapple, 64.5 ± 11.3%. The GI of glucose is taken as 100. The GI of pineapple was significantly lower than that of glucose (P < 0.05). Banana, pawpaw, and pineapple produced a similar postprandial glucose response. Measured portions of these fruits may be used as fruit exchanges with pineapple having the most favorable glycemic response.

Modulatory effect of pineapple peel extract on lipid peroxidation, catalase activity and hepatic biomarker levels in blood plasma of alcohol-induced oxidative stressed rats

PubMed Central

Okafor, OY; Erukainure, OL; Ajiboye, JA; Adejobi, RO; Owolabi, FO; Kosoko, SB

2011-01-01

Objective To investigate the ability of the methanolic extract of pineapple peel to modulate alcohol-induced lipid peroxidation, changes in catalase activities and hepatic biochemical marker levels in blood plasma. Methods Oxidative stress was induced by oral administration of ethanol (20% w/v) at a dosage of 5 mL/kg bw in rats. After 28 days of treatment, the rats were fasted overnight and sacrificed by cervical dislocation. Blood was collected with a 2 mL syringe by cardiac puncture and was centrifuged at 3 000 rpm for 10 min. The plasma was analyzed to evaluate malondialdehyde (MDA), catalase activity, aspartate aminotransferase (AST), alkaline phosphatase (ALP) and alanine aminotransferase (ALT) concentrations. Results Administration of alcohol caused a drastic increase (87.74%) in MDA level compared with the control. Pineapple peel extract significantly reduced the MDA level by 60.16% at 2.5 mL/kg bw. Rats fed alcohol only had the highest catalase activity, treatment with pineapple peel extract at 2.5 mL/kg bw however, reduced the activity. Increased AST, ALP and ALT activities were observed in rats fed alcohol only respectively, treatment with pineapple peel extract drastically reduced their activities. Conclusions The positive modulation of lipid peroxidation, catalase activities as well as hepatic biomarker levels of blood plasma by the methanolic extract of pineapple peels under alcohol-induced oxidative stress is an indication of its protective ability in the management of alcohol-induced toxicity. PMID:23569717

Testicular histology and blood testosterone levels of male rabbit after given concentrated diets containing calliandra leaf meal and pineapple peels

NASA Astrophysics Data System (ADS)

Setyawati, I.; Ermayanti, N. G. A. M.; Suarni, N. M. R.; Narayani, I.; Suaskara, I. B. M.

2018-03-01

Calliandra calothyrsus Meissn is one of a highly protein source of forage, however, it is not widely used for non ruminant feed because it contains antinutritional substances in the form of condensed tannin. Tannin can bind proteins, this tannin-protein complexes are difficult to be digested in the gastrointestinal tract, then will be defecated outside the body. To optimize the utilization of calliandra in the diet, pineapple peels were added as a source of protease (bromelain). Beside of waste utilization, it is expected that bromelain can degrade the tannin-protein complexes thereby reducing the negative effects of tannins. This research is a feeding experiment on male rabbit (Lepus sp), five weeks old. The diet formulation is prepared according to the standardized diet of the local rabbit. This experiment used a Completely Randomized Design (CRD) with four treatments i.e. control group which were only given commercial feed (R0), commercial feed contained 15% of Calliandra leaf meal and 30% of pineapple peel (R1), commercial feed contained 30% of Calliandra leaf meal and 30% of pineapple peel (R2), and commercial feed contained 45% of Calliandra and 30% of pineapple peel (R3). The treatment was done for three months. The results of this study on the reproduction of male rabbits showed that the increase of calliandra leaf meal level in a diet containing 30% of pineapple peels affected the testicular histology, and also decreased the diameter of seminiferous tubule and blood testosterone levels of male rabbits.

Alteration of Porcine Serum Albumin Levels in Pork Meat by Marination in Kiwi or Pineapple Juice and Subsequent Pan Broiling

PubMed Central

Moon, Sung-Sil; Kim, Il-Suk; Ham, Jun-Sang; Park, Beom-Young

2014-01-01

This study was conducted to evaluate the changes in porcine serum albumin (PSA), a major allergen, which occur when raw pork ham is marinated with kiwi or pineapple juice, and/or when the ham is pan broiled at 300℃ for 4 min after marination. In this study, raw pork ham was soaked for 4 h or 8 h in marinades containing commercial marinating sauce only, commercial marinating sauce and 7% kiwi juice, or commercial marinating sauce and 7% pineapple juice. When the meat was marinated and then pan-broiled, pork ham meat protein was significantly denatured and hydrolyzed, and the level of PSA in the meat was significantly reduced. The PSA contents of pork broiled without marination, pork that had been marinated in commercial marinating sauce alone, pork that had been marinated in commercial marinating sauce with kiwi juice, and pork that had been marinated in commercial marinating sauce with pineapple juice, were 95.4, 43.3, 14.3, and 5.4 ng/mL, respectively (p<0.05). Marinating with pineapple juice was more effective than marinating with kiwi juice; and marination for 8 h was more effective than marinating for 4 h. These results indicate that the level of PSA in pork ham is effectively reduced, when the meat is first marinated in sauces that contain kiwi or pineapple extracts for 8 h, rather than 4 h, and then cooked. Further study is needed to determine whether marinated pork meat reduces allergenicity in vivo, as well. PMID:26761177

Enzymatic, antimicrobial and toxicity studies of the aqueous extract of Ananas comosus (pineapple) crown leaf.

PubMed

Dutta, Sangita; Bhattacharyya, Debasish

2013-11-25

Various parts of the plant pineapple (Ananas comosus) are used in traditional medicine worldwide for treatment of a number of diseases and disorders. In folk medicine, pineapple leaf extract was used as an antimicrobial, vermicide, purgative, emmenagoogue, abortifacient, anti-oedema and anti-inflammatory agent. Compared to the fruit and stem extracts of pineapple, information about its leaf extract is limited. The potential of pineapple crown leaf extract as an ethno-medicine has been evaluated in terms of its enzymatic activities related to wound healing, antimicrobial property and toxicity. Major protein components of the extract were revealed by 2-D gel electrophoresis followed by MS/MS analysis. Zymography, DQ-gelatin assay were performed to demonstrate proteolytic, fibrinolytic, gelatinase and collagenase activities. DNase and RNase activities were revealed from agarose gel electrophoresis. Antimicrobial activity was evaluated spectrophotometrically from growth inhibition. Sprague-Dawley rat model was used to measure acute and sub-acute toxicity of the extract by analyzing blood markers. The extract contains several proteins that were clustered under native condition. Proteomic studies indicated presence of fruit bromelain as major protein constituent of the extract. It showed nonspecific protease activity, gelatinolytic, collagenase, fibrinolytic, acid and alkaline phosphatase, peroxidase, DNase and RNase activities along with considerable anti-microbial property. The leaf extract did not induce any toxicity in rats after oral administration of acute and sub-acute doses. Pineapple leaf extract is nontoxic, contains enzymes related to damage tissue repairing, wound healing and possibly prevents secondary infections from microbial organisms. © 2013 Elsevier Ireland Ltd. All rights reserved.

Isolation and characterization of a TERMINAL FLOWER 1 homolog from Prunus serotina Ehrh

Treesearch

Ying Wang; Paula M. Pijut

2013-01-01

Flowering control is one of the several strategies for gene containment of transgenic plants. TERMINAL FLOWER 1 (TFL1) is known to be involved in the transcriptional repression of genes for inflorescence development. Two TFL1 transcripts with different 3' UTR were cloned from black cherry (Prunus serotina...

Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene

USDA-ARS?s Scientific Manuscript database

The widespread use of atrazine and other s-triazine herbicides to control weeds in agricultural production fields has impacted surface and ground water in the United States and elsewhere. We previously reported the cloning, sequencing, and expression of six genes involved in the atrazine biodegradat...

[Plant transposable elements and their application in genetics and biotechnology].

PubMed

Ovcharenko, O O; Rudas, V A; Kuchuk, M V

2006-01-01

Data concerning plant transposable elements and their contribution to plant genome evolution are reviewed. Much attention is focused on utilization of transgenic plants as heterologous hosts of transposons for investigation of transposition mechanisms and gene cloning. Probable ways of the use of plant transposons as genetic tools in biotechnology are discussed.

RNAi construct of a P450 gene blocks an early step in Hemigossypolone and Gossypol synthesis in transgenic cotton plants

USDA-ARS?s Scientific Manuscript database

Naturally occurring terpenoid aldehydes from cotton such as gossypol, hemigossypolone, and heliocides, are important components of disease and herbivory resistance in cotton. These terpenoids are predominately found in the glands. Differential screening identified a P450 cDNA clone (GHC28) that on...

Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms.

PubMed

Ariotti, Nicholas; Hall, Thomas E; Rae, James; Ferguson, Charles; McMahon, Kerrie-Ann; Martel, Nick; Webb, Robyn E; Webb, Richard I; Teasdale, Rohan D; Parton, Robert G

2015-11-23

Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular cloning and characterization of a gene regulating flowering time from Alfalfa (Medicago sativa L.).

PubMed

Zhang, Tiejun; Chao, Yuehui; Kang, Junmei; Ding, Wang; Yang, Qingchuan

2013-07-01

Genes that regulate flowering time play crucial roles in plant development and biomass formation. Based on the cDNA sequence of Medicago truncatula (accession no. AY690425), the LFY gene of alfalfa was cloned. Sequence similarity analysis revealed high homology with FLO/LFY family genes of other plants. When fused to the green fluorescent protein, MsLFY protein was localized in the nucleus of onion (Allium cepa L.) epidermal cells. The RT-qPCR analysis of MsLFY expression patterns showed that the expression of MsLFY gene was at a low level in roots, stems, leaves and pods, and the expression level in floral buds was the highest. The expression of MsLFY was induced by GA3 and long photoperiod. Plant expression vector was constructed and transformed into Arabidopsis by the agrobacterium-mediated methods. PCR amplification with the transgenic Arabidopsis genome DNA indicated that MsLFY gene had integrated in Arabidopsis genome. Overexpression of MsLFY specifically caused early flowering under long day conditions compared with non-transgenic plants. These results indicated MsLFY played roles in promoting flowering time.

Modified expression of alternative oxidase in transgenic tomato and petunia affects the level of tomato spotted wilt virus resistance.

PubMed

Ma, Hao; Song, Congfeng; Borth, Wayne; Sether, Diane; Melzer, Michael; Hu, John

2011-10-20

Tomato spotted wilt virus (TSWV) has a very wide host range, and is transmitted in a persistent manner by several species of thrips. These characteristics make this virus difficult to control. We show here that the over-expression of the mitochondrial alternative oxidase (AOX) in tomato and petunia is related to TSWV resistance. The open reading frame and full-length sequence of the tomato AOX gene LeAox1au were cloned and introduced into tomato 'Healani' and petunia 'Sheer Madness' using Agrobacterium-mediated transformation. Highly expressed AOX transgenic tomato and petunia plants were selfed and transgenic R1 seedlings from 10 tomato lines and 12 petunia lines were used for bioassay. For each assayed line, 22 to 32 tomato R1 progeny in three replications and 39 to 128 petunia progeny in 13 replications were challenged with TSWV. Enzyme-Linked Immunosorbent Assays showed that the TSWV levels in transgenic tomato line FKT4-1 was significantly lower than that of wild-type controls after challenge with TSWV. In addition, transgenic petunia line FKP10 showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by TSWV. In all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower TSWV levels than non-transgenic plants after challenge with TSWV, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of AOX in tomato and petunia can affect the levels of TSWV resistance.

Transgenic cloned sheep overexpressing ovine toll-like receptor 4.

PubMed

Deng, Shoulong; Li, Guiguan; Zhang, Jinlong; Zhang, Xiaosheng; Cui, Maosheng; Guo, Yong; Liu, Guoshi; Li, Guangpeng; Feng, Jianzhong; Lian, Zhengxing

2013-07-01

An ovine fetal fibroblast cell line highly expressing TLR4 was established by inserting TLR4 into a reconstructive p3S-LoxP plasmid. Transgenic sheep overexpressing TLR4 were produced by transferring TLR4-transfected fetal fibroblasts into metaphase (M)II-stage enucleated oocytes (using SCNT). Because reconstructed embryos derived from MII-stage enucleated oocytes matured in vivo using a delayed-activated method had a higher pregnancy rate (18.52%) than that from MII-stage enucleated oocytes matured in vitro, the former procedure was used. Nine TLR4-transgenic live births were confirmed using polymerase chain reaction and Southern blot analysis. Increased expression of TLR4 at mRNA and protein levels in ear tissues of transgenic lambs were verified using reverse transcription polymerase chain reaction and immunohistochemistry, respectively. More toll-like receptor 4 protein was expressed by peripheral blood monocytes and/or macrophages collected from 3-month-old TLR4-transgenic than nontransgenic lambs at 0, 1, and 4 hours after lipopolysaccharide stimulation. Furthermore, interferon-γ and tumor necrosis factor α secreted by monocytes and/or macrophages of TLR4-transgenic lambs were significantly higher at 1 hour. Therefore, lipopolysaccharide-induced inflammatory responses from monocytes and/or macrophages occurred sooner in TLR4-transgenic lambs, consistent with an enhanced host immune response. In conclusion, transgenic sheep overexpressing TLR4 are a primary model to investigate the role of transgenic animals in disease resistance and have potential for breeding sheep with disease resistance. Copyright © 2013 Elsevier Inc. All rights reserved.

Modified expression of alternative oxidase in transgenic tomato and petunia affects the level of tomato spotted wilt virus resistance

PubMed Central

2011-01-01

Background Tomato spotted wilt virus (TSWV) has a very wide host range, and is transmitted in a persistent manner by several species of thrips. These characteristics make this virus difficult to control. We show here that the over-expression of the mitochondrial alternative oxidase (AOX) in tomato and petunia is related to TSWV resistance. Results The open reading frame and full-length sequence of the tomato AOX gene LeAox1au were cloned and introduced into tomato 'Healani' and petunia 'Sheer Madness' using Agrobacterium-mediated transformation. Highly expressed AOX transgenic tomato and petunia plants were selfed and transgenic R1 seedlings from 10 tomato lines and 12 petunia lines were used for bioassay. For each assayed line, 22 to 32 tomato R1 progeny in three replications and 39 to 128 petunia progeny in 13 replications were challenged with TSWV. Enzyme-Linked Immunosorbent Assays showed that the TSWV levels in transgenic tomato line FKT4-1 was significantly lower than that of wild-type controls after challenge with TSWV. In addition, transgenic petunia line FKP10 showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by TSWV. Conclusion In all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower TSWV levels than non-transgenic plants after challenge with TSWV, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of AOX in tomato and petunia can affect the levels of TSWV resistance. PMID:22014312

Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs

PubMed Central

Matsunari, Hitomi; Nagashima, Hiroshi; Watanabe, Masahito; Umeyama, Kazuhiro; Nakano, Kazuaki; Nagaya, Masaki; Kobayashi, Toshihiro; Yamaguchi, Tomoyuki; Sumazaki, Ryo; Herzenberg, Leonard A.; Nakauchi, Hiromitsu

2013-01-01

In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals. PMID:23431169

An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

PubMed

Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

2012-07-01

cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs.

PubMed

Matsunari, Hitomi; Nagashima, Hiroshi; Watanabe, Masahito; Umeyama, Kazuhiro; Nakano, Kazuaki; Nagaya, Masaki; Kobayashi, Toshihiro; Yamaguchi, Tomoyuki; Sumazaki, Ryo; Herzenberg, Leonard A; Nakauchi, Hiromitsu

2013-03-19

In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals.

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy1

PubMed Central

Gao, Xiaoqing; Zhou, Jie; Li, Jun; Zou, Xiaowei; Zhao, Jianhua; Li, Qingliang; Xia, Ran; Yang, Ruifang; Wang, Dekai; Zuo, Zhaoxue; Tu, Jumin; Tao, Yuezhi; Chen, Xiaoyun; Xie, Qi; Zhu, Zengrong

2015-01-01

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops. PMID:25371551

Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy.

PubMed

Gao, Xiaoqing; Zhou, Jie; Li, Jun; Zou, Xiaowei; Zhao, Jianhua; Li, Qingliang; Xia, Ran; Yang, Ruifang; Wang, Dekai; Zuo, Zhaoxue; Tu, Jumin; Tao, Yuezhi; Chen, Xiaoyun; Xie, Qi; Zhu, Zengrong; Qu, Shaohong

2015-01-01

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops. © 2015 American Society of Plant Biologists. All Rights Reserved.

76 FR 41248 - Fenamiphos; Notice of Receipt of Request to Amend Use Deletion and Product Cancellation Order

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-13

..., Maui Pineapple, to extend the deadline for sale and distribution of Nemacur 3 Emulsifiable Systemic... Agency received another request from Maui Pineapple to extend the deadline for sale and distribution of...

Effect of boron on fruit quality in pineapple

NASA Astrophysics Data System (ADS)

Wei, Changbin; Ma, Zhiling; Liu, Yuge; Qiao, Jian; Sun, Guangming

2018-04-01

Boron (B) is an important element for the plant. The aim of work was to study the effect of B on fruit quality of pineapple. The experiment was carried out with `Comte de paris' variety in pots. The results demonstrated that B had positive effect on fruit weight, TSS, the ratio of TSS/acidity, Vitamin C, the content of aroma volatile compounds. The B had no effect on the content of the three sugars (sucrose, fructose and glucose) and titrable acidity. There was a positive effect on fruit quality by application of B fertilizer in production of pineapple.

Applied K fertilizer use efficiency in pineapples grown on a tropical peat soil under residues removal.

PubMed

Ahmed, Osumanu H; Ahmad, Husni M H; Musa, Hanafi M; Rahim, Anuar A; Rastan, Syed Omar S

2005-01-21

In Malaysia, pineapples are grown on peat soils, but most K fertilizer recommendations do not take into account K loss through leaching. The objective of this study was to determine applied K use efficiency under a conventionally recommended fertilization regime in pineapple cultivation with residues removal. Results showed that K recovery from applied K fertilizer in pineapple cultivation on tropical peat soil was low, estimated at 28%. At a depth of 0-10 cm, there was a sharp decrease of soil total K, exchangeable K, and soil solution K days after planting (DAP) for plots with K fertilizer. This decline continued until the end of the study. Soil total, exchangeable, and solution K at the end of the study were generally lower than prior values before the study. There was no significant accumulation of K at depths of 10-25 and 25-45 cm. However, K concentrations throughout the study period were generally lower or equal to their initial status in the soil indicating leaching of the applied K and partly explained the low K recovery. Potassium losses through leaching in pineapple cultivation on tropical peat soils need to be considered in fertilizer recommendations for efficient recovery of applied K.

Electrodialytic removal of nitrate from pineapple juice: effect on selected physicochemical properties, amino acids, and aroma components of the juice.

PubMed

Ackarabanpojoue, Yuwadee; Chindapan, Nathamol; Yoovidhya, Tipaporn; Devahastin, Sakamon

2015-05-01

This study aimed at investigating the effect of nitrate removal from pineapple juice by electrodialysis (ED) on selected properties of the ED-treated juice. Single-strength pineapple juice with reduced pulp content was treated by ED to reduce the nitrate concentration to 15, 10, or 5 ppm. After ED, the removed pulp was added to the ED-treated juice and its properties, including electrical conductivity, acidity, pH, total soluble solids (TSS), color, amino acids, and selected aroma compounds, were determined and compared with those of the untreated juice. ED could reduce the nitrate content of 1 L of pineapple juice from an initial value of 50 ppm to less than 5 ppm within 30 min. A significant decrease in the electrical conductivity, acidity, pH, TSS, and yellowness, but a significant increase in the lightness, of the juice was observed upon ED. Concentrations of almost all amino acids of the ED-treated juice significantly decreased. The concentrations of 8 major compound contributors to the pineapple aroma also significantly decreased. Adding the pulp back to the ED-treated juice increased the amino acids concentrations; however, it led to a significant decrease in the concentrations of the aroma compounds. © 2015 Institute of Food Technologists®

Effects of high-pressure argon and nitrogen treatments on respiration, browning and antioxidant potential of minimally processed pineapples during shelf life.

PubMed

Wu, Zhi-shuang; Zhang, Min; Wang, Shao-jin

2012-08-30

High-pressure (HP) inert gas processing causes inert gas and water molecules to form clathrate hydrates that restrict intracellular water activity and enzymatic reactions. This technique can be used to preserve fruits and vegetables. In this study, minimally processed (MP) pineapples were treated with HP (∼10 MPa) argon (Ar) and nitrogen (N) for 20 min. The effects of these treatments on respiration, browning and antioxidant potential of MP pineapples were investigated after cutting and during 20 days of storage at 4 °C. Lower respiration rate and ethylene production were found in HP Ar- and HP N-treated samples compared with control samples. HP Ar and HP N treatments effectively reduced browning and loss of total phenols and ascorbic acid and maintained antioxidant capacity of MP pineapples. They did not cause a significant decline in tissue firmness or increase in juice leakage. HP Ar treatments had greater effects than HP N treatments on reduction of respiration rate and ethylene production and maintenance of phenolic compounds and DPPH(•) and ABTS(•+) radical-scavenging activities. Both HP Ar and HP N processing had beneficial effects on MP pineapples throughout 20 days of storage at 4 °C. Copyright © 2012 Society of Chemical Industry.

CLONING AND CHARACTERIZATION OF OSTEOCLAST PRECURSORS FROM THE RAW264.7 CELL LINE

PubMed Central

Cuetara, Bethany L. V.; Crotti, Tania N.; O'Donoghue, Anthony J.

2006-01-01

SUMMARY Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-κB (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell culture, which are poorly suited to molecular and transgene studies due to the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP) positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP positive multinuclear cells. Clones capable of forming large TRAP positive multinuclear cells also expressed β3 integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-κB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation. PMID:16948499

Isozyme variation in wild and cultivated pineapple

USDA-ARS?s Scientific Manuscript database

Isozyme variation was studied in 161 accessions of pineapple including four species of Ananas and one of Pseudananas. Six enzyme systems (ADH, GPI, PGM, SKDH, TPI, UGPP) involving seven putative loci revealed 35 electromorphs . Considerable variation exists within and between species of Ananas. Sixt...

The newt (Cynops pyrrhogaster) RPE65 promoter: molecular cloning, characterization and functional analysis.

PubMed

Casco-Robles, Martin Miguel; Miura, Tomoya; Chiba, Chikafumi

2015-06-01

The adult newt has the ability to regenerate the neural retina following injury, a process achieved primarily by the retinal pigment epithelium (RPE). To deliver exogenous genes to the RPE for genetic manipulation of regenerative events, we isolated the newt RPE65 promoter region by genome walking. First, we cloned the 2.8 kb RPE65 promoter from the newt, Cynops pyrrhogaster. Sequence analysis revealed several conserved regulatory elements described previously in mouse and human RPE65 promoters. Second, having previously established an I-SceI-mediated transgenic protocol for the newt, we used it here to examine the -657 bp proximal promoter of RPE65. The promoter assay used with F0 transgenic newts confirmed transgene expression of mCherry fluorescent protein in the RPE. Using bioinformatic tools and the TRANSFAC database, we identified a 340 bp CpG island located between -635 and -296 bp in the promoter; this region contains response elements for the microphthalmia-associated transcription factor known as MITF (CACGTG, CATGTG), and E-boxes (CANNTG). Sex-determining region box 9 (or SOX9) response element previously reported in the regulation of RPE genes (including RPE65) was also identified in the newt RPE65 promoter. Third, we identified DNA motif boxes in the newt RPE65 promoter that are conserved among other vertebrates. The newt RPE65 promoter is an invaluable tool for site-specific delivery of exogenous genes or genetic manipulation systems for the study of retinal regeneration in this animal.

Purification of a Jojoba Embryo Wax Synthase, Cloning of its cDNA, and Production of High Levels of Wax in Seeds of Transgenic Arabidopsis

PubMed Central

Lardizabal, Kathryn D.; Metz, James G.; Sakamoto, Tetsuo; Hutton, William C.; Pollard, Michael R.; Lassner, Michael W.

2000-01-01

Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a β-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. 13C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds. PMID:10712527

Sugarcane transgenics expressing MYB transcription factors show improved glucose release

DOE PAGES

Poovaiah, Charleson R.; Bewg, William P.; Lan, Wu; ...

2016-07-15

In this study, sugarcane, a tropical C4 perennial crop, is capable of producing 30-100 tons or more of biomass per hectare annually. The lignocellulosic residue remaining after sugar extraction is currently underutilized and can provide a significant source of biomass for the production of second-generation bioethanol. As a result, MYB31 and MYB42 were cloned from maize and expressed in sugarcane with and without the UTR sequences. The cloned sequences were 98 and 99 % identical to the published nucleotide sequences. The inclusion of the UTR sequences did not affect any of the parameters tested. There was little difference in plantmore » height and the number of internodes of the MYB-overexpressing sugarcane plants when compared with controls. MYB transgene expression determined by qPCR exhibited continued expression in young and maturing internodes. MYB31 downregulated more genes within the lignin biosynthetic pathway than MYB42. MYB31 and MYB42 expression resulted in decreased lignin content in some lines. All MYB42 plants further analyzed showed significant increases in glucose release by enzymatic hydrolysis in 72 h, whereas only two MYB31 plants released more glucose than control plants. This correlated directly with a significant decrease in acid-insoluble lignin. Soluble sucrose content of the MYB42 transgenic plants did not vary compared to control plants. In conclusion, this study demonstrates the use of MYB transcription factors to improve the production of bioethanol from sugarcane bagasse remaining after sugar extraction.« less

Sugarcane transgenics expressing MYB transcription factors show improved glucose release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poovaiah, Charleson R.; Bewg, William P.; Lan, Wu

In this study, sugarcane, a tropical C4 perennial crop, is capable of producing 30-100 tons or more of biomass per hectare annually. The lignocellulosic residue remaining after sugar extraction is currently underutilized and can provide a significant source of biomass for the production of second-generation bioethanol. As a result, MYB31 and MYB42 were cloned from maize and expressed in sugarcane with and without the UTR sequences. The cloned sequences were 98 and 99 % identical to the published nucleotide sequences. The inclusion of the UTR sequences did not affect any of the parameters tested. There was little difference in plantmore » height and the number of internodes of the MYB-overexpressing sugarcane plants when compared with controls. MYB transgene expression determined by qPCR exhibited continued expression in young and maturing internodes. MYB31 downregulated more genes within the lignin biosynthetic pathway than MYB42. MYB31 and MYB42 expression resulted in decreased lignin content in some lines. All MYB42 plants further analyzed showed significant increases in glucose release by enzymatic hydrolysis in 72 h, whereas only two MYB31 plants released more glucose than control plants. This correlated directly with a significant decrease in acid-insoluble lignin. Soluble sucrose content of the MYB42 transgenic plants did not vary compared to control plants. In conclusion, this study demonstrates the use of MYB transcription factors to improve the production of bioethanol from sugarcane bagasse remaining after sugar extraction.« less

Aroma profile and volatiles odor activity along gold cultivar pineapple flesh.

PubMed

Montero-Calderón, Marta; Rojas-Graü, María Alejandra; Martín-Belloso, Olga

2010-01-01

Physicochemical attributes, aroma profile, and odor contribution of pineapple flesh were studied for the top, middle, and bottom cross-sections cut along the central axis of Gold cultivar pineapple. Relationships between volatile and nonvolatile compounds were also studied. Aroma profile constituents were determined by headspace solid-phase microextraction at 30 °C, followed by gas chromatography/mass spectrometry analysis. A total of 20 volatile compounds were identified and quantified. Among them, esters were the major components which accounted for 90% of total extracted aroma. Methyl butanoate, methyl 2-methyl butanoate, and methyl hexanoate were the 3 most abundant components representing 74% of total volatiles in pineapple samples. Most odor active contributors were methyl and ethyl 2-methyl butanoate and 2,5-dimethyl 4-methoxy 3(2H)-furanone (mesifuran). Aroma profile components did not vary along the fruit, but volatile compounds content significantly varied (P < 0.05) along the fruit, from 7560 to 10910 μg/kg, from the top to the bottom cross-sections of the fruit, respectively. In addition, most odor-active volatiles concentration increased from the top to the bottom 3rd of the fruit, concurrently with soluble solids content (SSC) and titratable acidity (TA) differences attributed to fruitlets distinct degree of ripening. Large changes in SSC/TA ratio and volatiles content throughout the fruit found through this study are likely to provoke important differences among individual fresh-cut pineapple trays, compromising consumer perception and acceptance of the product. Such finding highlighted the need to include volatiles content and SSC/TA ratio and their variability along the fruit as selection criteria for pineapples to be processed and quality assessment of the fresh-cut fruit. © 2010 Institute of Food Technologists®

Development of Pineapple Microsatellite Markers and Germplasm Genetic Diversity Analysis

PubMed Central

Tong, Helin; Chen, You; Wang, Jingyi; Chen, Yeyuan; Sun, Guangming; He, Junhu; Wu, Yaoting

2013-01-01

Two methods were used to develop pineapple microsatellite markers. Genomic library-based SSR development: using selectively amplified microsatellite assay, 86 sequences were generated from pineapple genomic library. 91 (96.8%) of the 94 Simple Sequence Repeat (SSR) loci were dinucleotide repeats (39 AC/GT repeats and 52 GA/TC repeats, accounting for 42.9% and 57.1%, resp.), and the other three were mononucleotide repeats. Thirty-six pairs of SSR primers were designed; 24 of them generated clear bands of expected sizes, and 13 of them showed polymorphism. EST-based SSR development: 5659 pineapple EST sequences obtained from NCBI were analyzed; among 1397 nonredundant EST sequences, 843 were found containing 1110 SSR loci (217 of them contained more than one SSR locus). Frequency of SSRs in pineapple EST sequences is 1SSR/3.73 kb, and 44 types were found. Mononucleotide, dinucleotide, and trinucleotide repeats dominate, accounting for 95.6% in total. AG/CT and AGC/GCT were the dominant type of dinucleotide and trinucleotide repeats, accounting for 83.5% and 24.1%, respectively. Thirty pairs of primers were designed for each of randomly selected 30 sequences; 26 of them generated clear and reproducible bands, and 22 of them showed polymorphism. Eighteen pairs of primers obtained by the one or the other of the two methods above that showed polymorphism were selected to carry out germplasm genetic diversity analysis for 48 breeds of pineapple; similarity coefficients of these breeds were between 0.59 and 1.00, and they can be divided into four groups accordingly. Amplification products of five SSR markers were extracted and sequenced, corresponding repeat loci were found and locus mutations are mainly in copy number of repeats and base mutations in the flanking region. PMID:24024187

Insect resistance to sugar beet pests mediated by a Beta vulgaris proteinase inhibitor transgene

USDA-ARS?s Scientific Manuscript database

We transformed sugar beet (Beta vulgaris) hairy roots and Nicotiana benthamiana plants with a Beta vulgaris root gene (BvSTI) that codes for a serine proteinase inhibitor. BvSTI is a root gene cloned from the F1016 breeding line that has moderate levels of resistance to the sugar beet root maggot ...

The Effects of Predictability on Stress and Immune Function

DTIC Science & Technology

1993-06-24

number of cups ______________ _ Chocolate, cocoa, wine, beer/alcohol, decaffeinated coffee. Breads containing raisins, prunes, orange peel , banana or...pudding,. mince pie ••••• ) Banana , avocado, pineapple, canned figs, raisins, plums and prune •• Orange., orange juice, fruit cocktail with pineapple

Characterization of a polyhydroxyalkanoate obtained from pineapple peel waste using Ralsthonia eutropha.

PubMed

Vega-Castro, Oscar; Contreras-Calderon, Jose; León, Emilson; Segura, Almir; Arias, Mario; Pérez, León; Sobral, Paulo J A

2016-08-10

Agro-industrial waste can be the production source of biopolymers such as polyhydroxyalkanoates. The aim of this study was to produce and characterize Polyhydroxyalkanoates produced from pineapple peel waste fermentation processes. The methodology includes different pineapple peel waste fermentation conditions. The produced biopolymer was characterized using FTIR, GC-MS and NMR. The best fermentation condition for biopolymer production was obtained using pH 9, Carbon/Nitrogen 11, carbon/phosphorus 6 and fermentation time of 60h. FTIR analyzes showed PHB group characteristics, such as OH, CH and CO. In addition, GC-MS showed two monomers with 4 and 8 carbons, referred to PHB and PHBHV. H(1) NMR analysis showed 0.88-0.97 and 5.27ppm signals, corresponding to CH3 and CH, respectively. In conclusion, polyhydroxyalkanoate production from pineapple peels waste is an alternative for the treatment of waste generated in Colombia's fruit industry. Copyright © 2016 Elsevier B.V. All rights reserved.

Postharvest Exogenous Application of Abscisic Acid Reduces Internal Browning in Pineapple.

PubMed

Zhang, Qin; Liu, Yulong; He, Congcong; Zhu, Shijiang

2015-06-10

Internal browning (IB) is a postharvest physiological disorder causing economic losses in pineapple, but there is no effective control measure. In this study, postharvest application of 380 μM abscisic acid (ABA) reduced IB incidence by 23.4-86.3% and maintained quality in pineapple fruit. ABA reduced phenolic contents and polyphenol oxidase and phenylalanine ammonia lyase activities; increased catalase and peroxidase activities; and decreased O2(·-), H2O2, and malondialdehyde levels. This suggests ABA could control IB through inhibiting phenolics biosynthesis and oxidation and enhancing antioxidant capability. Furthermore, the efficacy of IB control by ABA was not obviously affected by tungstate, ABA biosynthesis inhibitor, nor by diphenylene iodonium, NADPH oxidase inhibitor, nor by lanthanum chloride, calcium channel blocker, suggesting that ABA is sufficient for controlling IB. This process might not involve H2O2 generation, but could involve the Ca(2+) channels activation. These results provide potential for developing effective measures for controlling IB in pineapple.

Ripening-dependent metabolic changes in the volatiles of pineapple (Ananas comosus (L.) Merr.) fruit: I. Characterization of pineapple aroma compounds by comprehensive two-dimensional gas chromatography-mass spectrometry.

PubMed

Steingass, Christof Björn; Carle, Reinhold; Schmarr, Hans-Georg

2015-03-01

Qualitative ripening-dependent changes of pineapple volatiles were studied via headspace solid-phase microextraction and analyzed by comprehensive two-dimensional gas chromatography quadrupole mass spectrometry (HS-SPME-GC×GC-qMS). Early green-ripe stage, post-harvest ripened, and green-ripe fruits at the end of their commercial shelf-life were compared to air-freighted pineapples harvested at full maturity. In total, more than 290 volatiles could be identified by mass spectrometry and their linear retention indices. The majority of compounds comprise esters (methyl and ethyl esters of saturated and unsaturated fatty acids, acetates), terpenes, alcohols, aldehydes, 2-ketones, free fatty acids, and miscellaneous γ- and δ-lactones. The structured separation space obtained by GC×GC allowed revealing various homologous series of compound classes as well as clustering of sesquiterpenes. Post-harvest ripening increased the diversity of the volatile profile compared to both early green-ripe maturity stages and on-plant ripened fruits.

Bromelain, a cysteine protease from pineapple (Ananas comosus) stem, is an inhibitor of fungal plant pathogens.

PubMed

López-García, B; Hernández, M; Segundo, B S

2012-07-01

This study aimed to evaluate the effect of bromelain, a cysteine protease isolated from pineapple (Ananas comosus), on growth of several agronomically important fungal pathogens. Purification of bromelain from pineapple stems was carried out by chromatography techniques, and its antimicrobial activity was tested against the fungal pathogens Fusarium verticillioides, Fusarium oxysporum and Fusarium proliferatum by broth microdilution assay. A concentration of 0.3 μmol l(-1) of bromelain was sufficient for 90% growth inhibition of F. verticillioides. The capability of bromelain to inhibit fungal growth is related to its proteolytic activity. The study demonstrates that stem bromelain exhibits a potent antifungal activity against phytopathogens and suggests its potential use as an effective agent for crop protection. The results support the use of a natural protease that accumulates at high levels in pineapple stems as alternative to the use of chemical fungicides for crop protection. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Ultrasound treatment on phenolic metabolism and antioxidant capacity of fresh-cut pineapple during cold storage.

PubMed

Yeoh, Wei Keat; Ali, Asgar

2017-02-01

Ultrasound treatment at different power output (0, 25 and 29W) and exposure time (10 and 15min) was used to investigate its effect on the phenolic metabolism enzymes, total phenolic content and antioxidant capacity of fresh-cut pineapple. Following ultrasound treatment at 25 and 29W, the activity of phenylalanine ammonia lyase (PAL) was increased significantly (P<0.05) by 2.0 and 1.9-fold, when compared to control. Meanwhile, both the activity of polyphenol oxidase (PPO) and polyphenol peroxidase (POD) in fresh-cut pineapple was significantly (P<0.05) lower than control upon subjected to ultrasound treatment. In the present study, induction of PAL was found to significantly (P<0.001) correlate with higher total phenolic content and thus higher antioxidant capacity in fresh-cut pineapple. Results suggest that hormetic dosage of ultrasound treatment can enhance the activity of PAL and total phenolic content and hence the total antioxidant capacity to encounter with oxidative stress. Copyright © 2016. Published by Elsevier Ltd.

Sustainable biobutanol production from pineapple waste by using Clostridium acetobutylicum B 527: Drying kinetics study.

PubMed

Khedkar, Manisha A; Nimbalkar, Pranhita R; Gaikwad, Shashank G; Chavan, Prakash V; Bankar, Sandip B

2017-02-01

Present investigation explores the use of pineapple peel, a food industry waste, for acetone-butanol-ethanol (ABE) production using Clostridium acetobutylicum B 527. Proximate analysis of pineapple peel shows that it contains 35% cellulose, 19% hemicellulose, and 16% lignin on dry basis. Drying experiments on pineapple peel waste were carried out in the temperature range of 60-120°C and experimental drying data was modeled using moisture diffusion control model to study its effect on ABE production. The production of ABE was further accomplished via acid hydrolysis, detoxification, and fermentation process. Maximum total sugar release obtained by using acid hydrolysis was 97g/L with 95-97% and 10-50% removal of phenolics and acetic acid, respectively during detoxification process. The maximum ABE titer obtained was 5.23g/L with 55.6% substrate consumption when samples dried at 120°C were used as a substrate (after detoxification). Copyright © 2016 Elsevier Ltd. All rights reserved.

A Phytophthora sojae cytoplasmic effector mediates disease resistance and abiotic stress tolerance in Nicotiana benthamiana.

PubMed

Zhang, Meixiang; Ahmed Rajput, Nasir; Shen, Danyu; Sun, Peng; Zeng, Wentao; Liu, Tingli; Juma Mafurah, Joseph; Dou, Daolong

2015-06-03

Each oomycete pathogen encodes a large number of effectors. Some effectors can be used in crop disease resistance breeding, such as to accelerate R gene cloning and utilisation. Since cytoplasmic effectors may cause acute physiological changes in host cells at very low concentrations, we assume that some of these effectors can serve as functional genes for transgenic plants. Here, we generated transgenic Nicotiana benthamiana plants that express a Phytophthora sojae CRN (crinkling and necrosis) effector, PsCRN115. We showed that its expression did not significantly affect the growth and development of N. benthamiana, but significantly improved disease resistance and tolerance to salt and drought stresses. Furthermore, we found that expression of heat-shock-protein and cytochrome-P450 encoding genes were unregulated in PsCRN115-transgenic N. benthamiana based on digital gene expression profiling analyses, suggesting the increased plant defence may be achieved by upregulation of these stress-related genes in transgenic plants. Thus, PsCRN115 may be used to improve plant tolerance to biotic and abiotic stresses.

The dynamics of long-term transgene expression in engrafted neural stem cells.

PubMed

Lee, Jean-Pyo; Tsai, David J; In Park, Kook; Harvey, Alan R; Snyder, Evan Y

2009-07-01

To assess the dynamics and confounding variables that influence transgene expression in neural stem cells (NSCs), we generated distinct NSC clones from the same pool of cells, carrying the same reporter gene transcribed from the same promoter, transduced by the same retroviral vector, and transplanted similarly at the same differentiation state, at the same time and location, into the brains of newborn mouse littermates, and monitored in parallel for over a year in vivo (without immunosuppression). Therefore, the sole variables were transgene chromosomal insertion site and copy number. We then adapted and optimized a technique that tests, at the single cell level, persistence of stem cell-mediated transgene expression in vivo based on correlating the presence of the transgene in a given NSC's nucleus (by fluorescence in situ hybridization [FISH]) with the frequency of that transgene's product within the same cell (by combined immunohistochemistry [IHC]). Under the above-stated conditions, insertion site is likely the most contributory variable dictating transgene downregulation in an NSC after 3 months in vivo. We also observed that this obstacle could be effectively and safely counteracted by simple serial infections (as few as three) inserting redundant copies of the transgene into the prospective donor NSC. (The preservation of normal growth control mechanisms and an absence of tumorigenic potential can be readily screened and ensured ex vivo prior to transplantation.) The combined FISH/IHC strategy employed here for monitoring the dynamics of transgene expression at the single cell level in vivo may be used for other types of therapeutic and housekeeping genes in endogenous and exogenous stem cells of many organs and lineages. Copyright 2009 Wiley-Liss, Inc.

Cell cycle distribution, cellular viability and mRNA expression of hGCase-gene-transfected cells in dairy goat.

PubMed

Zhang, Yan-Li; Wan, Yong-Jie; Wang, Zi-Yu; Qi, Wei-Wei; Zhou, Zheng-Rong; Huang, Rong; Wang, Feng

2010-05-07

Nuclear transfer using transgenic donor cells is an efficient way of generating transgenic goats, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. We have measured the efficiency of transfection with a plasmid containing hGCase (human lysosomal acid beta-glucosidase) gene into goat FFC (fetal-derived fibroblast cells), MEC (mammary epithelial cells) and AEFC (adult ear skin-derived fibroblast cells), and the characteristics of cell cycle, apoptosis and chromosome abnormalities after transfection. The expression of genes involved in imprinting [IGF2 (insulin-like growth factor 2), IGF2R (IGF2 receptor)], apoptosis (Bax), stress (heat-shock protein, Hsp70.1), cellular connections [Cx43 (connexin 43)] and DNA methylation [DNMT1 (DNA methyltransferase 1)] in transgenic fetal cells has been investigated. The hGCase transgene was successfully detected in the transfected cell lines, and chromosomal stability remained similar in FFC and transgenic FFC (70.9 compared with 66.8%), whereas a smaller percentage (P<0.05) of cells at G(0)/G(1) in the transgenic FFC, MEC and AEFC (T-FFC, T-MEC and T-AEFC), and higher percentage (P<0.05) of apoptotic cells in T-FFC than the non-transfected controls were detected by flow cytometric analysis. Among the genes tested, the relative expressions of IGF2, IGF2R and transcripts of Cx43 were significantly higher (P<0.05) in T-FFC compared with non-transfected FFC. These novel findings on gene expression in transgenic fetal cells may have certain implications in the biopharming industry and in our understanding the low efficiency of transgenic cloning.

Pineapple Waste Extract for Preventing Oxidation in Model Food Systems.

PubMed

Segovia Gómez, Francisco; Almajano Pablos, María Pilar

2016-07-01

Pineapple (Ananas comosus) is consumed in the form of chunks (canned), cubes, fruit salad, and also in juices, concentrates, and jams. In the processes to produce these products, the waste generated represents a high percentage of the total fruit. Some studies have shown that residues of certain fruits, such as pineapple, have the same antioxidant activity as the fruit pulp. So although these residues are discarded, they could be used as an alternative source of polyphenols, as natural antioxidants. This study is focused on the antioxidant activity of wastes obtained in the production of pineapple products and their application. The polyphenols' scavenging activity was determined by the oxygen radical antioxidant capacity assay. The antioxidant potential was determined in emulsions (o/w) and in muffins, where the primary oxidation products (by peroxide value, PV) and the secondary oxidation products (by thiobarbituric acid reactive substances) were analyzed. In addition the muffins were analyzed by means of a triangular sensory test. The PV method showed that pineapple waste extracts caused a reduction in oxidation products of 59% in emulsions and 91% in the muffins. The reduction in TBARs values for emulsions were 27% and for muffins were 51%. The triangular sensory test showed that the samples containing the extract were not distinguished from the control (α = 0.05). © 2016 Institute of Food Technologists®

Taxonomic structure of the yeasts and lactic acid bacteria microbiota of pineapple (Ananas comosus L. Merr.) and use of autochthonous starters for minimally processing.

PubMed

Di Cagno, Raffaella; Cardinali, Gainluigi; Minervini, Giovanna; Antonielli, Livio; Rizzello, Carlo Giuseppe; Ricciuti, Patrizia; Gobbetti, Marco

2010-05-01

Pichia guilliermondii was the only identified yeast in pineapple fruits. Lactobacillus plantarum and Lactobacillus rossiae were the main identified species of lactic acid bacteria. Typing of lactic acid bacteria differentiated isolates depending on the layers. L. plantarum 1OR12 and L. rossiae 2MR10 were selected within the lactic acid bacteria isolates based on the kinetics of growth and acidification. Five technological options, including minimal processing, were considered for pineapple: heating at 72 degrees C for 15 s (HP); spontaneous fermentation without (FP) or followed by heating (FHP), and fermentation by selected autochthonous L. plantarum 1OR12 and L. rossiae 2MR10 without (SP) or preceded by heating (HSP). After 30 days of storage at 4 degrees C, HSP and SP had a number of lactic acid bacteria 1000 to 1,000,000 times higher than the other processed pineapples. The number of yeasts was the lowest in HSP and SP. The Community Level Catabolic Profiles of processed pineapples indirectly confirmed the capacity of autochthonous starters to dominate during fermentation. HSP and SP also showed the highest antioxidant activity and firmness, the better preservation of the natural colours and were preferred for odour and overall acceptability. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Postharvest internal browning of pineapple fruit originates at the phloem.

PubMed

Luengwilai, Kietsuda; Beckles, Diane M; Siriphanich, Jingtair

2016-09-01

A typical symptom of postharvest chilling injury (PCI) in pineapple fruit (Ananas comosus (L.) Merr.) is internal browning (IB) near the fruit core. Since vascular bundles (VBs) are localized to this region, it was hypothesized that the VBs might be the site of IB. To test this, the anatomy and histochemistry of VBs during chilling stress in four pineapple cultivars with different levels of sensitivity to PCI were examined. Fruit were stored at 10°C for up to three weeks to stimulate translucency symptoms (TS; the initiation of IB). After three weeks of chilling exposure, the cultivars 'MD2' showed 0%, 'Pattavia' and 'Savee' showed 10-16%, and 'Trad Sri Thong' showed 100% TS and IB symptom. Scanning electron microscopy and in situ histochemical staining techniques that detect enzymes and substrates commonly associated with IB initiation were used in parallel. The TS of pineapple fruit coincided with the collapse of the phloem tissue. The VBs in the tissue where IB was initiated (i.e., the flesh adjacent to the core or F/C) had the highest activity of polyphenol oxidase, hydrogen peroxide, and phenolic compounds. The IB-resistant 'MD2' genotype had fewer VBs, but a greater proportion of sclerenchyma fibers (P<0.05) than did the susceptible 'Trad Sri Thong'. Based on these data, the first report of pineapple IB occurrence in the phloem was proposed. Copyright © 2016 Elsevier GmbH. All rights reserved.

Structural characteristics of pineapple pulp polysaccharides and their antitumor cell proliferation activities.

PubMed

Wang, Ling; Tang, De-Qiang; Kuang, Yu; Lin, Feng-Jiao; Su, Yu

2015-09-01

Pineapple has a delicious taste and good health benefits. Bioactive polysaccharides are important components of pineapple that might contribute to its health benefits. Since little structural information on these polysaccharides is currently available, the aim of this study was to investigate their structural characteristics and bioactivities. The polysaccharides of pineapple pulp were fractionated into three fractions (PAPs 1-3) by anion exchange chromatography. Their structural characteristics were first identified, including molecular weights and glycosidic linkages. The monosaccharide compositions were revealed as PAP 1 (Ara, Xyl, Man, Glc and Gal), PAP 2 (Rha, Ara, Xyl, Man, Glc and Gal) and PAP 3 (Rha, Ara, Xyl, Man and Gal). Nuclear magnetic resonance (NMR) spectra suggested that PAP 2 had a backbone of → 4)-α-d-Manp-(1 → 2,4)-α-d-Manp-(1 → with branches attached to O-4 of Manp. The NMR data of α-l-Araf-(1→, →3)-α-l-Araf-(1→, →4)-β-d-Galp-(1 → and → 4)-α-d-GalpAMe-(1 → were assigned. PAPs 1 and 2 showed significant antitumor cell proliferation activities against breast carcinoma cell line and strong antioxidant activities. The above findings indicated that PAPs 1-3 contributed much to the health benefits of pineapple. They could be used as health-beneficial food additives in functional foods. © 2015 Society of Chemical Industry.

Genome Enabled Discovery of Carbon Sequestration Genes in Poplar

DOE Office of Scientific and Technical Information (OSTI.GOV)

Filichkin, Sergei; Etherington, Elizabeth; Ma, Caiping

2007-02-22

The goals of the S.H. Strauss laboratory portion of 'Genome-enabled discovery of carbon sequestration genes in poplar' are (1) to explore the functions of candidate genes using Populus transformation by inserting genes provided by Oakridge National Laboratory (ORNL) and the University of Florida (UF) into poplar; (2) to expand the poplar transformation toolkit by developing transformation methods for important genotypes; and (3) to allow induced expression, and efficient gene suppression, in roots and other tissues. As part of the transformation improvement effort, OSU developed transformation protocols for Populus trichocarpa 'Nisqually-1' clone and an early flowering P. alba clone, 6K10. Completemore » descriptions of the transformation systems were published (Ma et. al. 2004, Meilan et. al 2004). Twenty-one 'Nisqually-1' and 622 6K10 transgenic plants were generated. To identify root predominant promoters, a set of three promoters were tested for their tissue-specific expression patterns in poplar and in Arabidopsis as a model system. A novel gene, ET304, was identified by analyzing a collection of poplar enhancer trap lines generated at OSU (Filichkin et. al 2006a, 2006b). Other promoters include the pGgMT1 root-predominant promoter from Casuarina glauca and the pAtPIN2 promoter from Arabidopsis root specific PIN2 gene. OSU tested two induction systems, alcohol- and estrogen-inducible, in multiple poplar transgenics. Ethanol proved to be the more efficient when tested in tissue culture and greenhouse conditions. Two estrogen-inducible systems were evaluated in transgenic Populus, neither of which functioned reliably in tissue culture conditions. GATEWAY-compatible plant binary vectors were designed to compare the silencing efficiency of homologous (direct) RNAi vs. heterologous (transitive) RNAi inverted repeats. A set of genes was targeted for post transcriptional silencing in the model Arabidopsis system; these include the floral meristem identity gene (APETALA1 or AP1), auxin response factor gene (ETTIN), the gene encoding transcriptional factor of WD40 family (TRANSPARENTTESTAGLABRA1 or TTG1), and the auxin efflux carrier (PIN-FORMED2 or PIN2) gene. More than 220 transgenic lines of the 1st, 2nd and 3rd generations were analyzed for RNAi suppression phenotypes (Filichkin et. al., manuscript submitted). A total of 108 constructs were supplied by ORNL, UF and OSU and used to generate over 1,881 PCR verified transgenic Populus and over 300 PCR verified transgenic Arabidopsis events. The Populus transgenics alone required Agrobacterium co-cultivations of 124.406 explants.« less

Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance, Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants

PubMed Central

Mishra, Manoj K.; Chaturvedi, Pankaj; Singh, Ruchi; Singh, Gaurav; Sharma, Lokendra K.; Pandey, Vibha; Kumari, Nishi; Misra, Pratibha

2013-01-01

Background Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant’s adaptation to abiotic stress. Methodology The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5. Results The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana. Conclusions Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases. PMID:23646175

21 CFR 146.185 - Pineapple juice.

Code of Federal Regulations, 2011 CFR

2011-04-01

... CONSUMPTION CANNED FRUIT JUICES Requirements for Specific Standardized Canned Fruit Juices and Beverages § 146... of shell, seeds, or other coarse or hard substances or excess pulp. It may be sweetened with any safe... pineapple juice (exclusive of added sugars) without added water shall not be less than 10.5° Brix as...

21 CFR 146.185 - Pineapple juice.

Code of Federal Regulations, 2012 CFR

2012-04-01

... CONSUMPTION CANNED FRUIT JUICES Requirements for Specific Standardized Canned Fruit Juices and Beverages § 146... of shell, seeds, or other coarse or hard substances or excess pulp. It may be sweetened with any safe... pineapple juice (exclusive of added sugars) without added water shall not be less than 10.5° Brix as...

21 CFR 146.185 - Pineapple juice.

Code of Federal Regulations, 2010 CFR

2010-04-01

... CONSUMPTION CANNED FRUIT JUICES Requirements for Specific Standardized Canned Fruit Juices and Beverages § 146... of shell, seeds, or other coarse or hard substances or excess pulp. It may be sweetened with any safe... pineapple juice (exclusive of added sugars) without added water shall not be less than 10.5° Brix as...

21 CFR 146.185 - Pineapple juice.

Code of Federal Regulations, 2013 CFR

2013-04-01

... CONSUMPTION CANNED FRUIT JUICES Requirements for Specific Standardized Canned Fruit Juices and Beverages § 146... of shell, seeds, or other coarse or hard substances or excess pulp. It may be sweetened with any safe... pineapple juice (exclusive of added sugars) without added water shall not be less than 10.5° Brix as...

21 CFR 146.185 - Pineapple juice.

Code of Federal Regulations, 2014 CFR

2014-04-01

... CONSUMPTION CANNED FRUIT JUICES Requirements for Specific Standardized Canned Fruit Juices and Beverages § 146... of shell, seeds, or other coarse or hard substances or excess pulp. It may be sweetened with any safe... pineapple juice (exclusive of added sugars) without added water shall not be less than 10.5° Brix as...

Droplet-vitrification and morphohistological studies of cryopreserved shoot tips of cultivated and wild pineapple genotypes

USDA-ARS?s Scientific Manuscript database

Germplasm conservation of pineapple [Ananas comosus (L.) Mer.] is crucial to preserve the genus’ genetic diversity to secure material for genetic improvement and to support innovative and new research. Long-term conservation is accomplished through cryopreservation that is done by storing cells or t...

The use of transgenic fruit trees as a resistance strategy for virus epidemics: the plum pox (sharka) model.

PubMed

Ravelonandro, M; Scorza, R; Callahan, A; Levy, L; Jacquet, C; Monsion, M; Damsteegt, V

2000-11-01

Sharka or plum pox, caused by Plum pox virus (PPV: genus Potyvirus; Family Potyviridae), is the most serious disease of Prunus. Most cultivated Prunus species are highly susceptible and conventional breeding has not produced highly resistant and commercially acceptable varieties. Success in developing virus-resistant herbaceous crops through genetic engineering led us to investigate this approach for resistance to PPV. Our programme aims to develop a biotechnological approach to PPV control that is effective and shown to be environmentally safe. The programme began with the cloning of the PPV coat protein (CP) gene and the development of a transformation system for plum (Prunus domestica). The CP construct was first tested in Nicotiana benthamiana in which it proved effective in producing transgenic plants with varying levels of CP expression. Some of these plants, particularly low PPV CP expressers, were resistant to PPV, or recovered from initial infection. Based on these results plum was transformed using the Agrobacterium tumefaciens system and both low and high PPV CP-expressing transgenic plum lines were obtained. These were inoculated with PPV by bud grafts in the greenhouse. Line C-5 proved to be highly resistant. It contained multiple copies of the insert, produced low levels of PPV CP mRNA, no detectable CP and the insert appeared to be methylated. These characteristics all suggest that the resistance of the C-5 clone is based on post-transcriptional gene silencing (PTGS). Field tests of C-5 and other transgenic lines in Poland, Romania and Spain have demonstrated that such trees when inoculated by bud-grafts allow a low level of PPV multiplication, from which they rapidly recover. C-5 plants exposed to natural infection for 3 years did not become infected, whereas control trees were infected in the first year. Hybrid plums having the C-5 PPV CP insert inherited from C-5 are virus-resistant, demonstrating the usefulness of C-5 as a parent in developing new PPV-resistant plum varieties. Research is in progress on the biorisks of PPV CP transgenic plants. Gene constructs that either produce no CP or CP that cannot be transmitted by aphids have been developed, tested in N. benthamiana and transferred to plum. Studies have begun on the potential for synergistic interactions between the PPV CP gene and the other common viruses of Prunus spp. In the future we will be participating in investigating the toxicity or/and the allergenicity of transgenic fruit products and, more importantly, transgenic lines will be developed that express transgenes only in vegetative parts of the plant and not in the fruit.

Hyper-reactive cloned mice generated by direct nuclear transfer of antigen-specific CD4+ T cells.

PubMed

Kaminuma, Osamu; Katayama, Kazufumi; Inoue, Kimiko; Saeki, Mayumi; Nishimura, Tomoe; Kitamura, Noriko; Shimo, Yusuke; Tofukuji, Soichi; Ishida, Satoru; Ogonuki, Narumi; Kamimura, Satoshi; Oikawa, Mami; Katoh, Shigeki; Mori, Akio; Shichijo, Michitaka; Hiroi, Takachika; Ogura, Atsuo

2017-06-01

T-cell receptor (TCR)-transgenic mice have been employed for evaluating antigen-response mechanisms, but their non-endogenous TCR might induce immune response differently than the physiologically expressed TCR Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen-specific CD4 + T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre-rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic-type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter-regulated antigen-specific TCR are a unique animal model with allergic predisposition for investigating CD4 + T-cell-mediated pathogenesis and cellular commitment in immune diseases. © 2017 The Authors.

Phytoremediation of arsenic from the contaminated soil using transgenic tobacco plants expressing ACR2 gene of Arabidopsis thaliana.

PubMed

Nahar, Noor; Rahman, Aminur; Nawani, Neelu N; Ghosh, Sibdas; Mandal, Abul

2017-11-01

We have cloned, characterized and transformed the AtACR2 gene (arsenic reductase 2) of Arabidopsis thaliana into the genome of tobacco (Nicotiana tabacum, var Sumsun). Our results revealed that the transgenic tobacco plants are more tolerant to arsenic than the wild type ones. These plants can grow on culture medium containing 200μM arsenate, whereas the wild type can barely survive under this condition. Furthermore, when exposed to 100μM arsenate for 35days the amount of arsenic accumulated in the shoots of transgenic plants was significantly lower (28μg/g d wt.) than that found in the shoots of non-transgenic controls (40μg/g d wt.). However, the arsenic content in the roots of transgenic plants was significantly higher (2400μg/g d. wt.) than that (2100μg/g d. wt.) observed in roots of wild type plants. We have demonstrated that Arabidopsis thaliana AtACR2 gene is a potential candidate for genetic engineering of plants to develop new crop cultivars that can be grown on arsenic contaminated fields to reduce arsenic content of the soil and can become a source of food containing no arsenic or exhibiting substantially reduced amount of this metalloid. Copyright © 2017 Elsevier GmbH. All rights reserved.

Ectopic expression of an apple cytochrome P450 gene MdCYPM1 negatively regulates plant photomorphogenesis and stress response in Arabidopsis.

PubMed

An, Jian-Ping; Li, Rui; Qu, Feng-Jia; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

2017-01-29

Cytochrome P450s play an important role in plant growth and are involved in multiple stresses response. However, little is known about the functions of cytochrome P450s in apple. Here, a Malus × domestica cytochrome P450 monooxygenase 1 gene, MdCYPM1, was identified and subsequently cloned from apple 'Gala' (Malus × domestica). To verify the functions of MdCYPM1, we generated transgenic Arabidopsis plants expressing the apple MdCYPM1 gene under the control of the Cauliflower mosaic virus 35S promoter. Four transgenic lines (#3, #5, #7 and #8) were selected for further study. The transgenic plants exhibited a series of skotomorphogenesis phenotypes relative to wild-type controls, such as reduction of the chlorophyll, anthocyanins content and hypocotyls elongation. In addition, overexpression of MdCYPM1 influenced auxin transport and flowering time in transgenic Arabidopsis. Furthermore, MdCYPM1 expression was induced by salt and mannitol treatments, and the transgenic plants were negatively regulated by salinity and osmotic stresses during germination. These results suggest that MdCYPM1 plays a vital role in plant growth and development. Copyright © 2017 Elsevier Inc. All rights reserved.

Xenografted islet cell clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice.

PubMed

Klymiuk, Nikolai; van Buerck, Lelia; Bähr, Andrea; Offers, Monika; Kessler, Barbara; Wuensch, Annegret; Kurome, Mayuko; Thormann, Michael; Lochner, Katharina; Nagashima, Hiroshi; Herbach, Nadja; Wanke, Rüdiger; Seissler, Jochen; Wolf, Eckhard

2012-06-01

Islet transplantation is a potential treatment for type 1 diabetes, but the shortage of donor organs limits its routine application. As potential donor animals, we generated transgenic pigs expressing LEA29Y, a high-affinity variant of the T-cell costimulation inhibitor CTLA-4Ig, under the control of the porcine insulin gene promoter. Neonatal islet cell clusters (ICCs) from INSLEA29Y transgenic (LEA-tg) pigs and wild-type controls were transplanted into streptozotocin-induced hyperglycemic NOD-scid IL2Rγ(null) mice. Cloned LEA-tg pigs are healthy and exhibit a strong β-cell-specific transgene expression. LEA-tg ICCs displayed the same potential to normalize glucose homeostasis as wild-type ICCs after transplantation. After adoptive transfer of human peripheral blood mononuclear cells, transplanted LEA-tg ICCs were completely protected from rejection, whereas reoccurrence of hyperglycemia was observed in 80% of mice transplanted with wild-type ICCs. In the current study, we provide the first proof-of-principle report on transgenic pigs with β-cell-specific expression of LEA29Y and their successful application as donors in a xenotransplantation model. This approach may represent a major step toward the development of a novel strategy for pig-to-human islet transplantation without side effects of systemic immunosuppression.

Agro-economic impact of cattle cloning.

PubMed

Faber, D C; Ferre, L B; Metzger, J; Robl, J M; Kasinathan, P

2004-01-01

The purpose of this paper is to review the economic and social implications of cloned cattle, their products, and their offspring as related to production agriculture. Cloning technology in cattle has several applications outside of traditional production agriculture. These applications can include bio-medical applications, such as the production of pharmaceuticals in the blood or milk of transgenic cattle. Cloning may also be useful in the production of research models. These models may or may not include genetic modifications. Uses in agriculture include many applications of the technology. These include making genetic copies of elite seed stock and prize winning show cattle. Other purposes may range from "insurance" to making copies of cattle that have sentimental value, similar to cloning of pets. Increased selection opportunities available with cloning may provide for improvement in genetic gain. The ultimate goal of cloning has often been envisioned as a system for producing quantity and uniformity of the perfect dairy cow. However, only if heritability were 100%, would clone mates have complete uniformity. Changes in the environment may have significant impact on the productivity and longevity of the resulting clones. Changes in consumer preferences and economic input costs may all change the definition of the perfect cow. The cost of producing such animals via cloning must be economically feasible to meet the intended applications. Present inefficiencies limit cloning opportunities to highly valued animals. Improvements are necessary to move the applications toward commercial application. Cloning has additional obstacles to conquer. Social and regulatory acceptance of cloning is paramount to its utilization in production agriculture. Regulatory acceptance will need to address the animal, its products, and its offspring. In summary, cloning is another tool in the animal biotechnology toolbox, which includes artificial insemination, sexing of semen, embryo sexing and in vitro fertilization. While it will not replace any of the above mentioned, its degree of utilization will depend on both improvement in efficiency as well as social and regulatory acceptance.

Pollination of pima pineapple cactus (Coryphantha sheeri var. robustispina): does pollen flow limit abundance of this endangered species?

Treesearch

Christopher J. McDonald; Guy R. McPherson

2005-01-01

Pima pineapple cactus (PPC) (Coryphantha sheeri var. robustispina), a federally listed endangered species, occurs throughout southeastern Arizona and has relatively low population densities. To determine whether pollination limits reproduction of PPC we used florescent dye to quantify pollen flow between individuals in a PPC...

Developing Single Nucleotide Polymorphism (SNP) markers for the identification of pineapple (Ananas comosus) germplasm

USDA-ARS?s Scientific Manuscript database

Pineapple (Ananas comosus [L.] Merr.) is the third most important tropical fruit in the world after banana and mango and a major agricultural commodity in Hawaii. As a crop with vegetative propagation, genetic redundancy is a major challenge for efficient genebank management and in breeding. Using E...

Droplet vitrification technique for cryopreservation of different pineapple (Ananas comosus L. Merrill) accessions

USDA-ARS?s Scientific Manuscript database

Germplasm conservation of pineapple is crucial to secure the genetic variability of the genus for breeding programs and supporting new research. Long-term conservation is done through cryopreservation, by storing cells or tissues at ultra-low temperature in liquid nitrogen (LN; -196°C) or in the LN ...

Natural Endophytic Occurrence of Acetobacter diazotrophicus in Pineapple Plants.

PubMed

Tapia-Hernández; Bustillos-Cristales; Jiménez-Salgado; Caballero-Mellado; Fuentes-Ramírez

2000-01-01

The presence of endophytic Acetobacter diazotrophicus was tested for pineapple plants (Ananas comosus [L.] Merr.) grown in the field. Diazotrophic bacteria were isolated from the inner tissues of surface sterilized roots, stems, and leaves of pineapple plants. Phenotypic tests permitted the selection of presumptive nitrogen-fixing A. diazotrophicus isolates. Restriction fragment length polymorphisms (RFLPs) of small subunit (SSU) rDNA using total DNA digested with endonuclease SphI and with endonuclease NcoI, hybridizations of RNA with an A. diazotrophicus large subunit (LSU) rRNA specific probe, as well as patterns in denaturing protein electrophoresis (SDS-PAGE) and multilocus enzyme tests allowed the identification of A. diazotrophicus isolates. High frequencies of isolation were obtained from propagative buds that had not been nitrogen-fertilized, and lower frequencies from 3-month-old plants that had been nitrogen-fertilized. No isolates were recovered from 5- to 7-month-old nitrogen-fertilized plants. All the A. diazotrophicus isolates recovered from pineapple plants belonged to the multilocus genotype which shows the most extensive distribution among all host species previously analyzed.

Light-induced alterations of pineapple (Ananas comosus [L.] Merr.) juice volatiles during accelerated ageing and mass spectrometric studies into their precursors.

PubMed

Steingass, Christof Björn; Glock, Mona Pia; Lieb, Veronika Maria; Carle, Reinhold

2017-10-01

Alterations of volatiles during accelerated light-induced ageing of pineapple juice were assessed by HS-SPME-GC-MS in a non-targeted profiling analysis over a 16-week period. Multivariate statistics permitted to reveal substantial chemical markers generally describing the effect of light storage. Volatiles generated comprised phenylpropenes, carbonyls, 2-methylthiophene, toluene, and furfural, while concentrations of methyl and ethyl esters, terpenes, and furanones decreased. In addition, the qualitative composition of phenolic compounds and glycoside-bound volatiles in selected samples was characterized by HPLC-DAD-ESI-MS n as well as HR-ESI-MS. The fresh juice contained unique pineapple metabolites such as S-p-coumaryl, S-coniferyl, S-sinapylglutathione, and structurally related derivatives. Among others, the presence of p-coumaroyl, feruloyl, and caffeoylisocitrate as well as three 4-hydroxy-2,5-dimethyl-3(2H)-furanone glycosides in pineapples could be substantiated by the HR-ESI-MS experiment. Mass spectrometric assignments of selected metabolites are presented, and putative linkages between volatiles and their precursors are established. Copyright © 2017 Elsevier Ltd. All rights reserved.

Survival of the endangered Pima pineapple cactus: Does clearing before prescribed fire alter survival postfire?

USGS Publications Warehouse

Thomas, Kathryn A.; Jarchow, Christopher; Crawford, Julie A.

2017-01-01

Federal land managers and ranchers often use prescribed fire as a tool to reduce invading woody plants within desert grasslands of the arid southwestern United States. Managers must evaluate the threat of the burn toward the health and survival of plants of concern including how preemptive clearing before prescribed fire might benefit these species. One example is the endangered Pima pineapple cactus (Coryphantha scheeri var. robustispina), a small hemispheric cactus of desert scrublands and grasslands of south-central Arizona and northern Sonora, Mexico. In 2014, we examined survival of Pima pineapple cactus documented in 2009 or 2010 within grasslands of Buenos Aires National Wildlife Refuge in Arizona. Of the 72 sites observed, 35 had no burn after documentation and 37 experienced prescribed fire. Refuge staff removed vegetation between 0.3 and 3.0 m from the cactus preburn. We found that Pima pineapple cacti in areas subjected to prescribed fire and with preemptive clearing had the same survival statistically as cacti from sites that were not burned.

Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity

PubMed Central

Schirmer, David; Grünewald, Thomas G. P.; Klar, Richard; Schmidt, Oxana; Wohlleber, Dirk; Rubío, Rebeca Alba; Uckert, Wolfgang; Thiel, Uwe; Bohne, Felix; Busch, Dirk H.; Krackhardt, Angela M.; Burdach, Stefan; Richter, Günther H. S.

2016-01-01

ABSTRACT Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8+ T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1130 peptide-driven stimulations with HLA-A*02:01+ dendritic cells (DC), allo-restricted HLA-A*02:01− CD8+ T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and β-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01− primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2−/−γc−/− mouse model. Initially generated transgenic T cells specifically recognized STEAP1130-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01+ ES lines more effectively than HLA-A*02:01− ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1130-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01+ ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors. PMID:27471654

Characterization of mutant tobacco mosaic virus coat protein that interferes with virus cell-to-cell movement.

PubMed

Bendahmane, Mohammed; Szecsi, Judit; Chen, Iju; Berg, R Howard; Beachy, Roger N

2002-03-19

Expression of tobacco mosaic virus (TMV) coat protein (CP) in plants confers resistance to infection by TMV and related tobamoviruses. Certain mutants of the CP (CP(T42W)) provide much greater levels of resistance than wild-type (wt) CP. In the present work, infection induced by RNA transcripts of TMV clones that contain wt CP or mutant CP(T42W) fused to the green fluorescent protein (GFP) (TMV-CP:GFP, TMV-CP(T42W):GFP) and clones harboring TMV movement protein (MP):GFP were followed in nontransgenic and transgenic tobacco BY-2 protoplasts and Nicotiana tabaccum Xanthi-nn plants that express wt CP or CP(T42W). On nontransgenic and wt CP transgenic plants, TMV-CP:GFP produced expanding, highly fluorescent disk-shaped areas. On plants expressing CP(T42W), infection by TMV-CP:GFP or TMV-MP:GFP-CP produced infection sites of smaller size that were characterized by low fluorescence, reflecting reduced levels of virus spread and reduced accumulation of both CP:GFP and MP:GFP. TMV-CP(T42W):GFP failed to produce visible infection sites on nontransgenic plants, yet produced normal infection sites on MP-transgenic plants that produce MP. TMV infection of transgenic BY-CP(T42W) protoplasts resulted in very low levels of MP accumulation, whereas on BY-CP protoplasts (containing wt CP), infection produced higher levels of MP than in nontransgenic BY-2 cells. The results suggest that wt CP has a positive effect on the production of MP, whereas the CP(T42W) has a negative effect on MP accumulation and/or function. This effect results in very high levels of resistance to TMV infection in plants containing CP(T42W). This report shows that the CP of a plant virus regulates production of the MP, and that a mutant CP interferes with MP accumulation and cell-to-cell movement of infection.

An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

PubMed

Char, Si Nian; Neelakandan, Anjanasree K; Nahampun, Hartinio; Frame, Bronwyn; Main, Marcy; Spalding, Martin H; Becraft, Philip W; Meyers, Blake C; Walbot, Virginia; Wang, Kan; Yang, Bing

2017-02-01

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T 0 transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T 1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Effect of pineapple waste powder on probiotic growth, antioxidant and antimutagenic activities of yogurt.

PubMed

Sah, B N P; Vasiljevic, T; McKechnie, S; Donkor, O N

2016-03-01

Although many fruit by-products are good sources of nutrients, little is known about their prebiotic potential. This research was aimed at establishing the prebiotic effect of pineapple wastes on probiotics including Lactobacillus (L.) acidophilus (ATCC® 4356™), L. casei (ATCC® 393™) and L. paracasei spp. paracasei (ATCC® BAA52™) and the subsequent release of antioxidant and antimutagenic peptides in yogurt during their growth. Oven- and freeze- dried peel and pomace were milled separately into powders and tested for prebiotic activities. The net probiotic growth (1.28-2.14 log cfu/g) in customized MRS broth containing the pineapple powders as a direct carbohydrate source was comparable to MRS broth containing glucose. The powders were also separately added to milk during the manufacturing of yogurt with or without probiotics. An increase (by 0.3-1.4 log cycle) in probiotic populations was observed in the yogurts as a consequence of pineapple powder supplementation. Crude water-soluble peptide extracts, prepared by high-speed centrifugation of the yogurts, displayed remarkable antioxidant activities assessed through in vitro assays, namely scavenging activity of 1,1-diphenyl-2-picrylhydrazyl radicals (IC50 = 0.37-0.19 mg/ml) and hydroxyl radicals (58.52-73.55 %). The peptide extracts also exhibited antimutagenic activities (18.60-32.72 %) as sodium azide inhibitor in the Salmonella mutagenicity test. Together, these results suggest that pineapple by-products exhibited prebiotic properties and could possibly be commercially applied in new functional food formulations.

Expression Patterns, Activities and Carbohydrate-Metabolizing Regulation of Sucrose Phosphate Synthase, Sucrose Synthase and Neutral Invertase in Pineapple Fruit during Development and Ripening

PubMed Central

Zhang, Xiu-Mei; Wang, Wei; Du, Li-Qing; Xie, Jiang-Hui; Yao, Yan-Li; Sun, Guang-Ming

2012-01-01

Differences in carbohydrate contents and metabolizing-enzyme activities were monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv. Comte de paris) during fruit development and ripening. Fructose and glucose of various sections in nearly equal amounts were the predominant sugars in the fruitlets, and had obvious differences until the fruit matured. The large rise of sucrose/hexose was accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a mechanism to increase fruit sink strength by increasing hexose concentrations. Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no. GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid sequences. Homology alignment reveals that the amino acid sequences contain some conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and Ac-ni also indicated distinct patterns related to sugar accumulation and composition of pineapple fruits. It suggests that differential expressions of multiple gene families are necessary for sugar metabolism in various parts and developmental stages of pineapple fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps by generating sugar signals to the cells in a temporally and spatially restricted fashion. PMID:22949808

Production of heterozygous alpha 1,3-galactosyltransferase (GGTA1) knock-out transgenic miniature pigs expressing human CD39.

PubMed

Choi, Kimyung; Shim, Joohyun; Ko, Nayoung; Eom, Heejong; Kim, Jiho; Lee, Jeong-Woong; Jin, Dong-Il; Kim, Hyunil

2017-04-01

Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig's cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig's cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.

Putative storage root specific promoters from cassava and yam: cloning and evaluation in transgenic carrots as a model system.

PubMed

Arango, Jacobo; Salazar, Bertha; Welsch, Ralf; Sarmiento, Felipe; Beyer, Peter; Al-Babili, Salim

2010-06-01

A prerequisite for biotechnological improvements of storage roots is the availability of tissue-specific promoters enabling high expression of transgenes. In this work, we cloned two genomic fragments, pMe1 and pDJ3S, controlling the expression of a gene with unknown function from cassava (Manihot esculenta) and of the storage protein dioscorin 3 small subunit gene from yam (Dioscorea japonica), respectively. Using beta-glucuronidase as a reporter, the activities of pMe1 and pDJ3S were evaluated in independent transgenic carrot lines and compared to the constitutive CaMV35S and the previously described cassava p15 promoters. Activities of pMe1 and pDJ3S in storage roots were assessed using quantitative GUS assays that showed pDJ3S as the most active one. To determine organ specificities, uidA transcript levels in leaves, stems and roots were measured by real-time RT-PCR analyses showing highest storage root specificity for pDJ3S. Root cross sections revealed that pMe1 was highly active in secondary xylem. In contrast, pDJ3S was active in all root tissues except for the central xylem. The expression patterns caused by the cassava p15 promoter in carrot storage roots were consistent with its previously described activities for the original storage organ. Our data demonstrate that the pDJ3S and, to a lesser extent, the pMe1 regulatory sequences represent feasible candidates to drive high and preferential expression of genes in carrot storage roots.

Modeling the Pineapple Express phenomenon via Multivariate Extreme Value Theory

NASA Astrophysics Data System (ADS)

Weller, G.; Cooley, D. S.

2011-12-01

The pineapple express (PE) phenomenon is responsible for producing extreme winter precipitation events in the coastal and mountainous regions of the western United States. Because the PE phenomenon is also associated with warm temperatures, the heavy precipitation and associated snowmelt can cause destructive flooding. In order to study impacts, it is important that regional climate models from NARCCAP are able to reproduce extreme precipitation events produced by PE. We define a daily precipitation quantity which captures the spatial extent and intensity of precipitation events produced by the PE phenomenon. We then use statistical extreme value theory to model the tail dependence of this quantity as seen in an observational data set and each of the six NARCCAP regional models driven by NCEP reanalysis. We find that most NCEP-driven NARCCAP models do exhibit tail dependence between daily model output and observations. Furthermore, we find that not all extreme precipitation events are pineapple express events, as identified by Dettinger et al. (2011). The synoptic-scale atmospheric processes that drive extreme precipitation events produced by PE have only recently begun to be examined. Much of the current work has focused on pattern recognition, rather than quantitative analysis. We use daily mean sea-level pressure (MSLP) fields from NCEP to develop a "pineapple express index" for extreme precipitation, which exhibits tail dependence with our observed precipitation quantity for pineapple express events. We build a statistical model that connects daily precipitation output from the WRFG model, daily MSLP fields from NCEP, and daily observed precipitation in the western US. Finally, we use this model to simulate future observed precipitation based on WRFG output driven by the CCSM model, and our pineapple express index derived from future CCSM output. Our aim is to use this model to develop a better understanding of the frequency and intensity of extreme precipitation events produced by PE under climate change.

Coupled social and ecological outcomes of agricultural intensification in Costa Rica and the future of biodiversity conservation in tropical agricultural regions

NASA Astrophysics Data System (ADS)

Sanfiorenzo, A. R.; Waits, L.; Finegan, B.; Shaver, I.; Chain Guadarrama, A.; Cleary, K.; Santiago-Garcia, R.; Hormel, L.; Vierling, L. A.; Bosque-Perez, N.; DeClerck, F.; Fagan, M. E.; Sibelet, N.

2016-12-01

Tropical ecosystem conversion to agriculture has caused widespread habitat loss and created fragmented landscapes composed of remnant forest patches embedded in a matrix of agricultural land uses. Non-traditional agricultural export (NTAE) crops such as pineapple are rapidly replacing multiuse landscapes characterized by a diverse matrix of pasture and smallholder crops with intensive, large-scale, monoculture plantations. Using an interdisciplinary approach, we examine the coupled social and ecological implications of agricultural intensification Guided by frameworks from political economy, landscape ecology and landscape genetics we: (1) describe the social and economic implications of pineapple expansion, specifically the concentration of land, labor and financial resources, (2) quantify pineapple cultivation's spatial characteristics, and (3) assess the effects of pineapple expansion on surrounding forest ecosystems, on the agricultural matrix and on biodiversity conservation. Our results indicate that pineapple production concentrates land, labor, and financial resources, which has a homogenizing effect on the agricultural economy in the study region. This constrains farm-based livelihoods, with larger implications for food security and agricultural diversity. Landscape ecology and genetics analyses further reveal how pineapple production simplifies and homogenizes the agricultural matrix between forest patches, which increase the genetic structure and reduce the genetic diversity of Symphonia globulifera a forest understory tree species. To offset the effects of agricultural intensification on social and environmental systems, we recommend developing landscape level land use planning capacity. Furthermore, agricultural and conservation policy reform is needed to promote landscape heterogeneity and economic diversity within the agricultural sector. Our interdisciplinary research provides a detailed examination of the social and ecological impacts of agricultural intensification in a tropical landscape, and offers recommendations for improvement relevant not only to our study region but to the many other tropical landscapes currently undergoing non-traditional agricultural export driven agricultural intensification.

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

PubMed Central

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-01-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure. PMID:26633465

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

PubMed

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-12-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

Construction of a multicontrol sterility system for a maize male-sterile line and hybrid seed production based on the ZmMs7 gene encoding a PHD-finger transcription factor.

PubMed

Zhang, Danfeng; Wu, Suowei; An, Xueli; Xie, Ke; Dong, Zhenying; Zhou, Yan; Xu, Liwen; Fang, Wen; Liu, Shensi; Liu, Shuangshuang; Zhu, Taotao; Li, Jinping; Rao, Liqun; Zhao, Jiuran; Wan, Xiangyuan

2018-02-01

Although hundreds of genetic male sterility (GMS) mutants have been identified in maize, few are commercially used due to a lack of effective methods to produce large quantities of pure male-sterile seeds. Here, we develop a multicontrol sterility (MCS) system based on the maize male sterility 7 (ms7) mutant and its wild-type Zea mays Male sterility 7 (ZmMs7) gene via a transgenic strategy, leading to the utilization of GMS in hybrid seed production. ZmMs7 is isolated by a map-based cloning approach and encodes a PHD-finger transcription factor orthologous to rice PTC1 and Arabidopsis MS1. The MCS transgenic maintainer lines are developed based on the ms7-6007 mutant transformed with MCS constructs containing the (i) ZmMs7 gene to restore fertility, (ii) α-amylase gene ZmAA and/or (iii) DNA adenine methylase gene Dam to devitalize transgenic pollen, (iv) red fluorescence protein gene DsRed2 or mCherry to mark transgenic seeds and (v) herbicide-resistant gene Bar for transgenic seed selection. Self-pollination of the MCS transgenic maintainer line produces transgenic red fluorescent seeds and nontransgenic normal colour seeds at a 1:1 ratio. Among them, all the fluorescent seeds are male fertile, but the seeds with a normal colour are male sterile. Cross-pollination of the transgenic plants to male-sterile plants propagates male-sterile seeds with high purity. Moreover, the transgene transmission rate through pollen of transgenic plants harbouring two pollen-disrupted genes is lower than that containing one pollen-disrupted gene. The MCS system has great potential to enhance the efficiency of maize male-sterile line propagation and commercial hybrid seed production. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Expression of the G72/G30 gene in transgenic mice induces behavioral changes

PubMed Central

Cheng, Lijun; Hattori, Eiji; Nakajima, Akira; Woehrle, Nancy S.; Opal, Mark D.; Zhang, Chunling; Grennan, Kay; Dulawa, Stephanie C.; Tang, Ya-Ping; Gershon, Elliot S.; Liu, Chunyu

2012-01-01

The G72/G30 gene complex is a candidate gene for schizophrenia and bipolar disorder. However, G72 and G30 mRNAs are expressed at very low levels in human brain, with only rare splicing forms observed. We report here G72/G30 expression profiles and behavioral changes in a G72/G30 transgenic mouse model. A human BAC clone containing the G72/G30 genomic region was used to establish the transgenic mouse model, on which gene expression studies, Western blot and behavioral tests were performed. Relative to their minimal expression in humans, G72 and G30 mRNAs were highly expressed in the transgenic mice, and had a more complex splicing pattern. The highest G72 transcript levels were found in testis, followed by cerebral cortex, with very low or undetectable levels in other tissues. No LG72 (the long putative isoform of G72) protein was detected in the transgenic mice. Whole-genome expression profiling identified 361 genes differentially-expressed in transgenic mice compared to wild-type, including genes previously implicated in neurological and psychological disorders. Relative to wild-type mice, the transgenic mice exhibited fewer stereotypic movements in the open field test, higher baseline startle responses in the course of the prepulse inhibition test, and lower hedonic responses in the sucrose preference test. The transcriptome profile changes and multiple mouse behavioral effects suggest that the G72 gene may play a role in modulating behaviors relevant to psychiatric disorders. PMID:23337943

Complete chloroplast genome sequence of MD-2 pineapple and its comparative analysis among nine other plants from the subclass Commelinidae.

PubMed

Redwan, R M; Saidin, A; Kumar, S V

2015-08-12

Pineapple (Ananas comosus var. comosus) is known as the king of fruits for its crown and is the third most important tropical fruit after banana and citrus. The plant, which is indigenous to South America, is the most important species in the Bromeliaceae family and is largely traded for fresh fruit consumption. Here, we report the complete chloroplast sequence of the MD-2 pineapple that was sequenced using the PacBio sequencing technology. In this study, the high error rate of PacBio long sequence reads of A. comosus's total genomic DNA were improved by leveraging on the high accuracy but short Illumina reads for error-correction via the latest error correction module from Novocraft. Error corrected long PacBio reads were assembled by using a single tool to produce a contig representing the pineapple chloroplast genome. The genome of 159,636 bp in length is featured with the conserved quadripartite structure of chloroplast containing a large single copy region (LSC) with a size of 87,482 bp, a small single copy region (SSC) with a size of 18,622 bp and two inverted repeat regions (IRA and IRB) each with the size of 26,766 bp. Overall, the genome contained 117 unique coding regions and 30 were repeated in the IR region with its genes contents, structure and arrangement similar to its sister taxon, Typha latifolia. A total of 35 repeats structure were detected in both the coding and non-coding regions with a majority being tandem repeats. In addition, 205 SSRs were detected in the genome with six protein-coding genes contained more than two SSRs. Comparative chloroplast genomes from the subclass Commelinidae revealed a conservative protein coding gene albeit located in a highly divergence region. Analysis of selection pressure on protein-coding genes using Ka/Ks ratio showed significant positive selection exerted on the rps7 gene of the pineapple chloroplast with P less than 0.05. Phylogenetic analysis confirmed the recent taxonomical relation among the member of commelinids which support the monophyly relationship between Arecales and Dasypogonaceae and between Zingiberales to the Poales, which includes the A. comosus. The complete sequence of the chloroplast of pineapple provides insights to the divergence of genic chloroplast sequences from the members of the subclass Commelinidae. The complete pineapple chloroplast will serve as a reference for in-depth taxonomical studies in the Bromeliaceae family when more species under the family are sequenced in the future. The genetic sequence information will also make feasible other molecular applications of the pineapple chloroplast for plant genetic improvement.

The expression pattern of the Picea glauca Defensin 1 promoter is maintained in Arabidopsis thaliana, indicating the conservation of signalling pathways between angiosperms and gymnosperms.

PubMed

Germain, Hugo; Lachance, Denis; Pelletier, Gervais; Fossdal, Carl Gunnar; Solheim, Halvor; Séguin, Armand

2012-01-01

A 1149 bp genomic fragment corresponding to the 5' non-coding region of the PgD1 (Picea glauca Defensin 1) gene was cloned, characterized, and compared with all Arabidopsis thaliana defensin promoters. The cloned fragment was found to contain several motifs specific to defence or hormonal response, including a motif involved in the methyl jasmonate reponse, a fungal elicitor responsive element, and TC-rich repeat cis-acting element involved in defence and stress responsiveness. A functional analysis of the PgD1 promoter was performed using the uidA (GUS) reporter system in stably transformed Arabidopsis and white spruce plants. The PgD1 promoter was responsive to jasmonic acid (JA), to infection by fungus and to wounding. In transgenic spruce embryos, GUS staining was clearly restricted to the shoot apical meristem. In Arabidopsis, faint GUS coloration was observed in leaves and flowers and a strong blue colour was observed in guard cells and trichomes. Transgenic Arabidopsis plants expressing the PgD1::GUS construct were also infiltrated with the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. It caused a suppression of defensin expression probably resulting from the antagonistic relationship between the pathogen-stimulated salicylic acid pathway and the jasmonic acid pathway. It is therefore concluded that the PgD1 promoter fragment cloned appears to contain most if not all the elements for proper PgD1 expression and that these elements are also recognized in Arabidopsis despite the phylogenetic and evolutionary differences that separates them.

Tissue-specific expression of human CD4 in transgenic mice.

PubMed Central

Gillespie, F P; Doros, L; Vitale, J; Blackwell, C; Gosselin, J; Snyder, B W; Wadsworth, S C

1993-01-01

The gene for the human CD4 glycoprotein, which serves as the receptor for human immunodeficiency virus type 1, along with approximately 23 kb of sequence upstream of the translational start site, was cloned. The ability of 5' flanking sequences to direct tissue-specific expression was tested in cell culture and in transgenic mice. A 5' flanking region of 6 kb was able to direct transcription of the CD4 gene in NIH 3T3 cells but did not result in detectable expression in the murine T-cell line EL4 or in four lines of transgenic mice. A larger 5' flanking region of approximately 23 kb directed high-level CD4 transcription in the murine T-cell line EL4 and in three independent lines of transgenic mice. Human CD4 expression in all tissues analyzed was tightly correlated with murine CD4 expression; the highest levels of human CD4 RNA expression were found in the thymus and spleen, with relatively low levels detected in other tissues. Expression of human CD4 protein in peripheral blood mononuclear cells was examined by flow cytometry in these transgenic animals and found to be restricted to the murine CD4+ subset of lymphocytes. Human CD4 protein, detected with an anti-human CD4 monoclonal antibody, was present on the surface of 45 to 50% of the peripheral blood mononuclear cells from all transgenic lines. Images PMID:8474453

High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro.

PubMed

Song, Shaozheng; Ge, Xin; Cheng, Yaobin; Lu, Rui; Zhang, Ting; Yu, Baoli; Ji, Xueqiao; Qi, Zhengqiang; Rong, Yao; Yuan, Yuguo; Cheng, Yong

2016-08-01

The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2-630 µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits.

Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant.

PubMed Central

Tsuchimoto, S; van der Krol, A R; Chua, N H

1993-01-01

We cloned a MADS-box gene, pMADS3, from Petunia hybrida, which shows high sequence homology to the Arabidopsis AGAMOUS and Antirrhinum PLENA. pMADS3 is expressed exclusively in stamens and carpels of wild-type petunia plants. In the petunia mutant blind, which shows homeotic conversions of corolla limbs into antheroid structures with pollen grains and small parts of sepals into carpelloid tissue, pMADS3 is expressed in all floral organs as well as in leaves. Ectopic expression of pMADS3 in transgenic petunia leads to phenocopies of the blind mutant, i.e., the formation of antheroid structures on limbs and carpelloid tissue on sepals. Transgenic tobacco plants that overexpress pMADS3 exhibit an even more severe phenotype, with the sepals forming a carpel-like structure encasing the interior floral organs. Our results identify BLIND as a negative regulator of pMADS3, which specifies stamens and carpels during petunia flower development. PMID:8104573

Functional characterization of CCR in birch (Betula platyphylla × Betula pendula) through overexpression and suppression analysis.

PubMed

Zhang, Wenbo; Wei, Rui; Chen, Su; Jiang, Jing; Li, Huiyu; Huang, Haijiao; Yang, Guang; Wang, Shuo; Wei, Hairong; Liu, Guifeng

2015-06-01

We cloned a Cinnamoyl-CoA Reductase gene (BpCCR1) from an apical meristem and first internode of Betula platyphylla and characterized its functions in lignin biosynthesis, wood formation and tree growth through transgenic approaches. We generated overexpression and suppression transgenic lines and analyzed them in comparison with the wild-type in terms of lignin content, anatomical characteristics, height and biomass. We found that BpCCR1 overexpression could increase lignin content up to 14.6%, and its underexpression decreased lignin content by 6.3%. Surprisingly, modification of BpCCR1 expression led to conspicuous changes in wood characteristics, including xylem vessel number and arrangement, and secondary wall thickness. The growth of transgenic trees in terms of height was also significantly influenced by the modification of BpCCR1 genes. We discuss the functions of BpCCR1 in the context of a phylogenetic tree built with CCR genes from multiple species. © 2014 Scandinavian Plant Physiology Society.

In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos.

PubMed

Gui, Tao; Zhang, Meiling; Chen, Jianwen; Zhang, Yuanliang; Zhou, Naru; Zhang, Yu; Tao, Jia; Sui, Liucai; Li, Yunsheng; Liu, Ya; Zhang, Xiaorong; Zhang, Yunhai

2012-08-01

A vector expressing human lysozyme (pBC1-hLYZ-GFP-Neo) was evaluated for gene and protein expression following liposome-mediated transformation of C-127 mouse mammary cancer cells. Cultures of G418-resistant clones were harvested 24-72 h after induction with prolactin, insulin and hydrocortisone. Target gene expression was analyzed by RT-PCR and Western blot and recombinant human lysozyme (rhLYZ) bacteriostatic activity was also evaluated. The hLYZ gene was correctly transcribed and translated in C-127 cells and hLYZ inhibited gram-positive bacterial growth, indicating the potential of this expression vector for development of a mammary gland bioreactor in goats. Guanzhong dairy goat skin fibroblasts transfected with pBC1-hLYZ-GFP-Neo were used to construct a goat embryo transgenically expressing rhLYZ by somatic nuclear transplantation with a blastocyst rate of 9.0 ± 2.8 %. These data establish the basis for cultivation of mastitis-resistant hLYZ transgenic goats.

Improvement of cloning efficiency in minipigs using post-thawed donor cells treated with roscovitine.

PubMed

Hwang, Seongsoo; Oh, Keon Bong; Kwon, Dae-Jin; Ock, Sun-A; Lee, Jeong-Woong; Im, Gi-Sun; Lee, Sung-Soo; Lee, Kichoon; Park, Jin-Ki

2013-11-01

Massachusetts General Hospital miniature pigs (MGH minipigs) have been established for organ transplantation studies across the homozygous major histocompatibility complex, but cloning efficiency of MGH minipigs is extremely low. This study was designed to increase the productivity of MGH minipigs by nuclear transfer of post-thaw donor cells after 1 h co-incubation with roscovitine. The MGH minipig cells were genetically modified with GT KO (alpha1,3-galactosyltransferase knock-out) and hCD46 KI (human CD46 knock-in) and used as donor cells. The GT KO/hCD46 KI donor cells were cultured for either 3 days (control group) or 1 h after thawing with 15 μM roscovitine (experimental group) prior to the nuclear transfer. The relative percentage of the transgenic donor cells that entered into G0/G1 was 93.7 % (±2.54). This was different from the donor cells cultured for 1 h with the roscovitine-treated group (84.6 % ±4.6) (P < 0.05) and without roscovitine (78.6 % ±5.5) (P < 0.01), respectively. The pregnancy rate and delivery rate in the roscovitine group (8/12 and 6/8, respectively) were significantly higher (P < 0.01) than those in the control group (6/19 and 3/6, respectively). In the experimental group, 12 GT KO/hCD46 KI transgenic minipigs were successfully generated, and five minipigs among them survived for more than 6 months so far. The recipient-based individual cloning efficiency ranged from 0.74 to 2.54 %. In conclusion, gene-modified donor cells can be used for cloning of MGH minipigs if the cells are post-thawed and treated with roscovitine for 1 h prior to nuclear transfer.

Mechanism of internal browning of pineapple: The role of gibberellins catabolism gene (AcGA2ox) and GAs

PubMed Central

Zhang, Qin; Rao, Xiuwen; Zhang, Lubin; He, Congcong; Yang, Fang; Zhu, Shijiang

2016-01-01

Internal browning (IB), a physiological disorder (PD) that causes severe losses in harvested pineapple, can be induced by exogenous gibberellins (GAs). Over the years, studies have focused on roles of Gibberellin 2-oxidase (GA2oxs), the major GAs catabolic enzyme in plants, in the regulation of changes in morphology or biomass. However, whether GA2oxs could regulate PD has not been reported. Here, a full-length AcGA2ox cDNA was isolated from pineapple, with the putative protein sharing 23.59% to 72.92% identity with GA2oxs from five other plants. Pineapples stored at 5 °C stayed intact, while those stored at 20 °C showed severe IB. Storage at 5 °C enhanced AcGA2ox expression and decreased levels of a GAs (GA4) ‘compared with storage at 20 °C. However, at 20 °C, exogenous application of abscisic acid (ABA) significantly suppressed IB. ABA simultaneously upregulated AcGA2ox and reduced GA4. Ectopic expression of AcGA2ox in Arabidopsis resulted in reduced GA4, lower seed germination, and shorter hypocotyls and roots, all of which were restored by exogenous GA4/7. Moreover, in pineapple, GA4/7 upregulated polyphenol oxidase, while storage at 5 °C and ABA downregulated it. These results strongly suggest the involvement of AcGA2ox in regulation of GAs levels and a role of AcGA2ox in regulating IB. PMID:27982026

Comparative content of total polyphenols and dietary fiber in tropical fruits and persimmon.

PubMed

Gorinstein, S; Zemser, M; Haruenkit, R; Chuthakorn, R; Grauer, F; Martin-Belloso, O; Trakhtenberg, S

1999-06-01

Recent studies have shown that dietary fiber and polyphenols of vegetables and fruits improve lipid metabolism and prevent the oxidation of low density lipoprotein cholesterol (LDL-C), which hinder the development of atherosclerosis. The goal of this study was to measure the total polyphenol and dietary fiber contents of some tropical fruits (i.e., pineapple, wax apple, rambutan, lichi, guava, and mango) and compare the results to the content of these substances in the better characterized persimmon. It was found that lichi, guava, and ripe mango (cv. Keaw) have 3.35, 4.95, and 6.25 mg of total polyphenols in 100 g fresh fruit, respectively. This is significantly higher than in persimmon, pineapple, wax apple, mature green mango, and rambutan [P < 0.0005 for pineapple (Smooth Cayene variant), wax apple, persimmon, rambutan, mature green mango (cv. Keaw); the value of P < 0.001 is found only for pineapple (Phuket, Queen variant)]. The same relationship was observed for the contents of gallic acid and of dietary fiber. It can be supposed that among the studied fruit, lichi, guava, and ripe mango may be preferable for dietary prevention of atherosclerosis.

Utilization of composite membrane polyethyleneglycol-polystyrene-cellulose acetate from pineapple leaf fibers in lowering levels of methyl orange batik waste

NASA Astrophysics Data System (ADS)

Delsy, E. V. Y.; Irmanto; Kazanah, F. N.

2017-02-01

Pineapple leaves are agricultural waste from the pineapple that the fibers can be utilized as raw material in cellulose acetate membranes. First, made pineapple leaf fibers into pulp and then converted into cellulose acetate by acetylation process in four stages consisting of activation, acetylation, hydrolysis and purification. Cellulose acetate then used as the raw material to manufacture composite membrane with addition of polystyrene and poly (ethylene glycol) as porogen. Composite membrane is made using phase inversion method with dichloromethane-acetone as a solvent. The result of FTIR analysis (Fourier transform infra-red) showed that the absorption of the carbonyl group (C=O) is at 1643.10 cm-1 and acetyl group (C-O ) at 1227.01 cm-1, with a molecular weight of 8.05 x 104 g/mol and the contents (rate) of acetyl is 37.31%. PS-PEG-CA composite membrane had also been characterized by measuring the water flux values and its application to decrease methyl orange content (level) in batik waste. The results showed that the water flux value is of 25.62 L/(m2.hour), and the decrease percentage of methyl orange content in batik waste is 71.53%.

Isolation of bacterial cellulose nanocrystalline from pineapple peel waste: Optimization of acid concentration in the hydrolysis method

NASA Astrophysics Data System (ADS)

Anwar, Budiman; Rosyid, Nurul Huda; Effendi, Devi Bentia; Nandiyanto, Asep Bayu Dani; Mudzakir, Ahmad; Hidayat, Topik

2016-02-01

Isolation of needle-shaped bacterial cellulose nanocrystalline with a diameter of 16-64 nm, a fiber length of 258-806 nm, and a degree of crystallinity of 64% from pineapple peel waste using an acid hydrolysis process was investigated. Experimental showed that selective concentration of acid played important roles in isolating the bacterial cellulose nanocrystalline from the cellulose source. To achieve the successful isolation of bacterial cellulose nanocrystalline, various acid concentrations were tested. To confirm the effect of acid concentration on the successful isolation process, the reaction conditions were fixed at a temperature of 50°C, a hydrolysis time of 30 minutes, and a bacterial cellulose-to-acid ratio of 1:50. Pineapple peel waste was used as a model for a cellulose source because to the best of our knowledge, there is no report on the use of this raw material for producing bacterial cellulose nanocrystalline. In fact, this material can be used as an alternative for ecofriendly and cost-free cellulose sources. Therefore, understanding in how to isolate bacterial cellulose nanocrystalline from pineapple peel waste has the potential for large-scale production of inexpensive cellulose nanocrystalline.

Development of Transgenic Minipigs with Expression of Antimorphic Human Cryptochrome 1

PubMed Central

Liu, Chunxin; Bolund, Lars; Vajta, Gábor; Dou, Hongwei; Yang, Wenxian; Xu, Ying; Luan, Jing; Wang, Jun; Yang, Huanming; Staunstrup, Nicklas Heine; Du, Yutao

2013-01-01

Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3–20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1AP). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders. PMID:24146819

Interpreting a sequenced genome: toward a cosmid transgenic library of Caenorhabditis elegans.

PubMed

Janke, D L; Schein, J E; Ha, T; Franz, N W; O'Neil, N J; Vatcher, G P; Stewart, H I; Kuervers, L M; Baillie, D L; Rose, A M

1997-10-01

We have generated a library of transgenic Caenorhabditis elegans strains that carry sequenced cosmids from the genome of the nematode. Each strain carries an extrachromosomal array containing a single cosmid, sequenced by the C. elegans Genome Sequencing Consortium, and a dominate Rol-6 marker. More than 500 transgenic strains representing 250 cosmids have been constructed. Collectively, these strains contain approximately 8 Mb of sequence data, or approximately 8% of the C. elegans genome. The transgenic strains are being used to rescue mutant phenotypes, resulting in a high-resolution map alignment of the genetic, physical, and DNA sequence maps of the nematode. We have chosen the region of chromosome III deleted by sDf127 and not covered by the duplication sDp8(III;I) as a starting point for a systematic correlation of mutant phenotypes with nucleotide sequence. In this defined region, we have identified 10 new essential genes whose mutant phenotypes range from developmental arrest at early larva, to maternal effect lethal. To date, 8 of these 10 essential genes have been rescued. In this region, these rescues represent approximately 10% of the genes predicted by GENEFINDER and considerably enhance the map alignment. Furthermore, this alignment facilitates future efforts to physically position and clone other genes in the region. [Updated information about the Transgenic Library is available via the Internet at http://darwin.mbb.sfu.ca/imbb/dbaillie/cos mid.html.

Effect of Alkali treatments on physical and Mechanical strength of Pineapple leaf fibres

NASA Astrophysics Data System (ADS)

Asim, M.; Jawaid, M.; Abdan, K.; Nasir, M.

2018-01-01

Pineapple leaf fibre (PALF) is a waste material of pineapple plants. PALF is abundant in amount for industrial purpose, cheap, easily available, high specific strength and stiffness. PALF is contributing a sustainable development in bio-composites as reinforcement material. However, natural fibres are not fully compatible with matrix due to hydrophilic in nature. To enhance the compatibility with matrix, fibres are modified its surface to make good interfacial bonding with matrix. In this research, PALF is treated with 3% and 6% concentration of NaOH for 3h, 6h 9h, and 12h soaking time. Surface modification of fibres was investigated by using scanning electron microscopy. Single fibre test and diameter of PALF fibres were evaluated the effects of NaOH treatments.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, Robert L.

1998-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .epsilon.N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, R.L.

1998-03-03

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) {epsilon}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 5 figs.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, R.L.

1999-02-02

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS){sup {epsilon}}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 8 figs.

A Mutation of the Prdm9 Mouse Hybrid Sterility Gene Carried by a Transgene.

PubMed

Mihola, O; Trachtulec, Z

2017-01-01

PRDM9 is a protein with histone-3-methyltransferase activity, which specifies the sites of meiotic recombination in mammals. Deficiency of the Prdm9 gene in the laboratory mouse results in complete arrest of the meiotic prophase of both sexes. Moreover, the combination of certain PRDM9 alleles from different mouse subspecies causes hybrid sterility, e.g., the male-specific meiotic arrest found in the (PWD/Ph × C57BL/6J)F1 animals. The fertility of all these mice can be rescued using a Prdm9-containing transgene. Here we characterized a transgene made from the clone RP24-346I22 that was expected to encompass the entire Prdm9 gene. Both (PWD/Ph × C57BL/6J)F1 intersubspecific hybrid males and Prdm9-deficient laboratory mice of both sexes carrying this transgene remained sterile, suggesting that Prdm9 inactivation occurred in the Tg(RP24-346I22) transgenics. Indeed, comparative qRT-PCR analysis of testicular RNAs from transgene-positive versus negative animals revealed similar expression levels of Prdm9 mRNAs from the exons encoding the C-terminal part of the protein but elevated expression from the regions coding for the N-terminus of PRDM9, indicating that the transgenic carries a new null Prdm9 allele. Two naturally occurring alternative Prdm9 mRNA isoforms were overexpressed in Tg(RP24-346I22), one formed via splicing to a 3'-terminal exon consisting of short interspersed element B2 and one isoform including an alternative internal exon of 28 base pairs. However, the overexpression of these alternative transcripts was apparently insufficient for Prdm9 function or for increasing the fertility of the hybrid males.

[Cloning and sequence analysis of tomato fruit-specific E8 promoter from Lycopersicon esculentum (Zhongshu No.5)].

PubMed

Zhou, Xiao-hong; Chen, Xiao-guang; Zhang, Xiao-dong; Wang, Ya-nan; Li, Lin; Xi, Jia-fei; Hu, Jian-jun

2003-01-01

To obtain the gene encoding tomato fruit-specific E8 promoter therefore to prepare for exogenous gene transcription and expression in transgenic tomato fruit. The cotyledons of tomato Lycopersicon esculentum (Zhongshu No.5) were collected for extracting the genomic DNA of this plant. The fruit-specific E81.1 and E82.2 promoter DNA were then amplified by PCR, the product of which was subcloned into pGEM-T vector. After identification by restriction enzymes, the recombinant T-vectors were subjected to sequence analysis. The fragments of the promoter as amplified by PCR were of predicted length. Digestion with Xba I and Hind III /BamH I proved correct insertion of the target fragments with expected length into the recombinant T vectors. As indicated by homology analysis, the resultant tomato fruit-specific E8 promoter was highly conservative, and E82.2 promoter of Zhongshu No.5, with GenBank submission number of AF515784, proved to share 99% homology with E82.2 promoter of Zhongshu No.5 Cherry as reported by Deikman J. Tomato fruit-specific E8 promoter of Zhongshu No.5 has been successfully cloned, thus making possible the subsequent research in oral vaccine of transgenic tomato.

A remorin gene SiREM6, the target gene of SiARDP, from foxtail millet (Setaria italica) promotes high salt tolerance in transgenic Arabidopsis.

PubMed

Yue, Jing; Li, Cong; Liu, Yuwei; Yu, Jingjuan

2014-01-01

Remorin proteins (REMs) form a plant-specific protein family, with some REMs being responsive to abiotic stress. However, the precise functions of REMs in abiotic stress tolerance are not clear. In this study, we identified 11 remorin genes from foxtail millet (Setaria italica) and cloned a remorin gene, SiREM6, for further investigation. The transcript level of SiREM6 was increased by high salt stress, low temperature stress and abscisic acid (ABA) treatment, but not by drought stress. The potential oligomerization of SiREM6 was examined by negative staining electron microscopy. The overexpression of SiREM6 improved high salt stress tolerance in transgenic Arabidopsis at the germination and seedling stages as revealed by germination rate, survival rate, relative electrolyte leakage and proline content. The SiREM6 promoter contains two dehydration responsive elements (DRE) and one ABA responsive element (ABRE). An ABA responsive DRE-binding transcription factor, SiARDP, and an ABRE-binding transcription factor, SiAREB1, were cloned from foxtail millet. SiARDP could physically bind to the DREs, but SiAREB1 could not. These results revealed that SiREM6 is a target gene of SiARDP and plays a critical role in high salt stress tolerance.

Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys.

PubMed

Petersen, Björn; Ramackers, Wolf; Lucas-Hahn, Andrea; Lemme, Erika; Hassel, Petra; Queisser, Anna-Lisa; Herrmann, Doris; Barg-Kues, Brigitte; Carnwath, Joseph W; Klose, Johannes; Tiede, Andreas; Friedrich, Lars; Baars, Wiebke; Schwinzer, Reinhard; Winkler, Michael; Niemann, Heiner

2011-01-01

The major immunological hurdle to successful porcine-to-human xenotransplantation is the acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and perturbation of coagulation. Heme oxygenase-1 (HO-1) and its derivatives have anti-apoptotic, anti-inflammatory effects and protect against reactive oxygen species, rendering HO-1 a promising molecule to control AVR. Here, we report the production and characterization of pigs transgenic for human heme oxygenase-1 (hHO-1) and demonstrate significant protection in porcine kidneys against xenograft rejection in ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells (PAEC) in a TNF-α-mediated apoptosis assay. Transgenic and non-transgenic PAEC were tested in a TNF-α-mediated apoptosis assay. Expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) was measured by real-time PCR. hHO-1 transgenic porcine kidneys were perfused with pooled and diluted human AB blood in an ex vivo perfusion circuit. MHC class-II up-regulation after induction with IFN-γ was compared between wild-type and hHO-1 transgenic PAEC. Cloned hHO-1 transgenic pigs expressed hHO-1 in heart, kidney, liver, and in cultured ECs and fibroblasts. hHO-1 transgenic PAEC were protected against TNF-α-mediated apoptosis. Real-time PCR revealed reduced expression of adhesion molecules like ICAM-1, VCAM-1, and E-selectin. These effects could be abrogated by the incubation of transgenic PAECs with the specific HO-1 inhibitor zinc protoporphorine IX (Zn(II)PPIX, 20 μm). IFN-γ induced up-regulation of MHC class-II molecules was significantly reduced in PAECs from hHO-1 transgenic pigs. hHO-1 transgenic porcine kidneys could successfully be perfused with diluted human AB-pooled blood for a maximum of 240 min (with and without C1 inh), while in wild-type kidneys, blood flow ceased after ∼60 min. Elevated levels of d-Dimer and TAT were detected, but no significant consumption of fibrinogen and antithrombin was determined. Microthrombi could not be detected histologically. These results are encouraging and warrant further studies on the biological function of heme oxygenase-I expression in hHO-1 transgenic pigs in the context of xenotransplantation. © 2011 John Wiley & Sons A/S.

Transgenic Tobacco Plants Overexpressing a Grass PpEXP1 Gene Exhibit Enhanced Tolerance to Heat Stress

PubMed Central

Xu, Qian; Xu, Xiao; Shi, Yang; Xu, Jichen; Huang, Bingru

2014-01-01

Heat stress is a detrimental abiotic stress limiting the growth of many plant species and is associated with various cellular and physiological damages. Expansins are a family of proteins which are known to play roles in regulating cell wall elongation and expansion, as well as other growth and developmental processes. The in vitro roles of expansins regulating plant heat tolerance are not well understood. The objectives of this study were to isolate and clone an expansin gene in a perennial grass species (Poa pratensis) and to determine whether over-expression of expansin may improve plant heat tolerance. Tobacco (Nicotiana tabacum) was used as the model plant for gene transformation and an expansin gene PpEXP1 from Poa pratensis was cloned. Sequence analysis showed PpEXP1 belonged to α-expansins and was closely related to two expansin genes in other perennial grass species (Festuca pratensis and Agrostis stolonifera) as well as Triticum aestivum, Oryza sativa, and Brachypodium distachyon. Transgenic tobacco plants over-expressing PpEXP1 were generated through Agrobacterium-mediated transformation. Under heat stress (42°C) in growth chambers, transgenic tobacco plants over-expressing the PpEXP1 gene exhibited a less structural damage to cells, lower electrolyte leakage, lower levels of membrane lipid peroxidation, and lower content of hydrogen peroxide, as well as higher chlorophyll content, net photosynthetic rate, relative water content, activity of antioxidant enzyme, and seed germination rates, compared to the wild-type plants. These results demonstrated the positive roles of PpEXP1 in enhancing plant tolerance to heat stress and the possibility of using expansins for genetic modification of cool-season perennial grasses in the development of heat-tolerant germplasm and cultivars. PMID:25003197

Identification and suppression of the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L.

PubMed Central

Marita, Jane M; Hatfield, Ronald D; Rancour, David M; Frost, Kenneth E

2014-01-01

Grasses, such as Zea mays L. (maize), contain relatively high levels of p-coumarates (pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase (pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full-length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls. PMID:24654730

Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions.

PubMed

Graser, R T; DiLorenzo, T P; Wang, F; Christianson, G J; Chapman, H D; Roopenian, D C; Nathenson, S G; Serreze, D V

2000-04-01

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic beta cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic beta cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4alphabeta Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted beta cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.

Identification and suppression of the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L.

PubMed

Marita, Jane M; Hatfield, Ronald D; Rancour, David M; Frost, Kenneth E

2014-06-01

Grasses, such as Zea mays L. (maize), contain relatively high levels of p-coumarates (pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase (pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full-length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

77 FR 24459 - Voluntary Termination of Foreign-Trade Subzone 9D, Maui Pineapple Company, Ltd., Kahului, Maui, HI

Federal Register 2010, 2011, 2012, 2013, 2014

2012-04-24

... DEPARTMENT OF COMMERCE Foreign-Trade Zones Board [Order No. 1821] Voluntary Termination of Foreign-Trade Subzone 9D, Maui Pineapple Company, Ltd., Kahului, Maui, HI Pursuant to its authority under the Foreign-Trade Zones Act of June 18, 1934, as amended (19 U.S.C. 81a-81u), and the Foreign-Trade Zones...

40 CFR 407.62 - Effluent limitations guidelines representing the degree of effluent reduction attainable by the...

Code of Federal Regulations, 2013 CFR

2013-07-01

....18 0.15 Pineapples 2.13 1.33 0.96 Plums 0.69 0.42 0.29 Raisins 0.43 0.28 0.21 Strawberries 1.79 1.06....33 0.25 Pineapples 3.85 2.76 1.81 Plums 1.24 0.87 0.54 Raisins 0.78 0.57 0.39 Strawberries 3.19 2.20...

40 CFR 407.62 - Effluent limitations guidelines representing the degree of effluent reduction attainable by the...

Code of Federal Regulations, 2014 CFR

2014-07-01

....18 0.15 Pineapples 2.13 1.33 0.96 Plums 0.69 0.42 0.29 Raisins 0.43 0.28 0.21 Strawberries 1.79 1.06....33 0.25 Pineapples 3.85 2.76 1.81 Plums 1.24 0.87 0.54 Raisins 0.78 0.57 0.39 Strawberries 3.19 2.20...

40 CFR 407.62 - Effluent limitations guidelines representing the degree of effluent reduction attainable by the...

Code of Federal Regulations, 2011 CFR

2011-07-01

....18 0.15 Pineapples 2.13 1.33 0.96 Plums 0.69 0.42 0.29 Raisins 0.43 0.28 0.21 Strawberries 1.79 1.06....33 0.25 Pineapples 3.85 2.76 1.81 Plums 1.24 0.87 0.54 Raisins 0.78 0.57 0.39 Strawberries 3.19 2.20...

40 CFR 407.62 - Effluent limitations guidelines representing the degree of effluent reduction attainable by the...

Code of Federal Regulations, 2012 CFR

2012-07-01

....18 0.15 Pineapples 2.13 1.33 0.96 Plums 0.69 0.42 0.29 Raisins 0.43 0.28 0.21 Strawberries 1.79 1.06....33 0.25 Pineapples 3.85 2.76 1.81 Plums 1.24 0.87 0.54 Raisins 0.78 0.57 0.39 Strawberries 3.19 2.20...

40 CFR 407.62 - Effluent limitations guidelines representing the degree of effluent reduction attainable by the...

Code of Federal Regulations, 2010 CFR

2010-07-01

....18 0.15 Pineapples 2.13 1.33 0.96 Plums 0.69 0.42 0.29 Raisins 0.43 0.28 0.21 Strawberries 1.79 1.06....33 0.25 Pineapples 3.85 2.76 1.81 Plums 1.24 0.87 0.54 Raisins 0.78 0.57 0.39 Strawberries 3.19 2.20...

Quantification of allyl hexanoate in pineapple beverages and yogurts as a case study to characterise a source of uncertainty in dietary exposure assessment to flavouring substances.

PubMed

Raffo, A; D'Aloise, A; Magrì, A D; Leclercq, C

2012-01-01

One source of uncertainty in the estimation of dietary exposure to flavouring substances is the uncertainty in the occurrence and concentration levels of these substances naturally present or added to foodstuffs. The aim of this study was to assess the variability of concentration levels of allyl hexanoate, considered as a case study, in two main food categories to which it is often added: pineapple juice-based beverages and yogurts containing pineapple. Thirty-four beverages and 29 yogurts, with pineapple fruit or juice and added flavourings declared as ingredients on the package, were purchased from the local market (in Rome) and analysed. Analytical methods based on the stir bar sorptive extraction (SBSE) technique for the isolation of the target analyte, and on GC-MS analysis for final determination, were developed for the two food categories. In beverages, allyl hexanoate concentrations ranged from less than 0.01 to 16.71 mg l(-1), whereas in yogurts they ranged from 0.02 to 89.41 mg kg(-1). Average concentrations in beverages and yogurts with pineapple as the main fruit ingredient (1.91 mg l(-1) for beverages, 9.61 mg kg(-1) for yogurts) were in fair agreement with average use level data reported from industry surveys for the relevant food categories (4.5 and 6.0 mg kg(-1), respectively). Within the group of yogurts a single product was found to contain a level of allyl hexanoate more than 10-fold higher than the average reported use level. The screening techniques developed by the European Food Safety Authority (EFSA) using use level data provided by industry gave estimates of exposure that were of the same order of magnitude as the estimates obtained for regular consumers who would be loyal to the pineapple yogurt and beverage products containing the highest observed concentration of the substance of interest. In this specific case the uncertainty in the results obtained with the use of standard screening techniques for exposure assessment based on industry reported use levels is low.

Nuclear transfer to study the nuclear reprogramming of human stem cells.

PubMed

Saito, Shigeo; Sawai, Ken; Murayama, Yoshinobu; Fukuda, Keiichi; Yokoyama, Kazunari

2008-01-01

Research of stem cells will enable us to understand the development and function of tissues and organs in mammals. The ability to induce regeneration of new tissues from embryonic stem (ES) cells derived from cloned blastocysts via nuclear transfer can be expected in the not-too-distant future. The fact that there is no way except nuclear cloning for the return of differentiated cells to undifferentiated cells remains an interesting problem to be solved. We describe protocols for the production of cloned calves from bovine ES cells to study nuclear reprogramming ability of stem cells. The frequency of term pregnancies for blastocysts from ES cells is higher than those of early pregnancies and maintained pregnancies after nuclear transfer with bovine somatic cells. We also describe protocols for gene introduction into bovine ES cells in vitro, particularly the human leukocyte antigens (HLA). Bovine ES cells provide a powerful tool for the generation of transgenic clonal offspring. This technique, when perfected for humans, may be critical for neural stem cell transplantation.

Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight.

PubMed

Karmakar, Subhasis; Molla, Kutubuddin Ali; Chanda, Palas K; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

2016-01-01

Green tissue-specific simultaneous overexpression of two defense-related genes ( OsCHI11 & OsOXO4 ) in rice leads to significant resistance against sheath blight pathogen ( R. solani ) without distressing any agronomically important traits. Overexpressing two defense-related genes (OsOXO4 and OsCHI11) cloned from rice is effective at enhancing resistance against sheath blight caused by Rhizoctonia solani. These genes were expressed under the control of two different green tissue-specific promoters, viz. maize phosphoenolpyruvate carboxylase gene promoter, PEPC, and rice cis-acting 544-bp DNA element, immediately upstream of the D54O translational start site, P D54O-544 . Putative T0 transgenic rice plants were screened by PCR and integration of genes was confirmed by Southern hybridization of progeny (T1) rice plants. Successful expression of OsOXO4 and OsCHI11 in all tested plants was confirmed. Expression of PR genes increased significantly following pathogen infection in overexpressing transgenic plants. Following infection, transgenic plants exhibited elevated hydrogen peroxide levels, significant changes in activity of ROS scavenging enzymes and reduced membrane damage when compared to their wild-type counterpart. In a Rhizoctonia solani toxin assay, a detached leaf inoculation test and an in vivo plant bioassay, transgenic plants showed a significant reduction in disease symptoms in comparison to non-transgenic control plants. This is the first report of overexpression of two different PR genes driven by two green tissue-specific promoters providing enhanced sheath blight resistance in transgenic rice.

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice.

PubMed Central

Soares, V da C; Gubits, R M; Feigelson, P; Costantini, F

1987-01-01

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene. Images PMID:2446121

Comparative landscape genetics of two frugivorous bats in a biological corridor undergoing agricultural intensification.

PubMed

Cleary, Katherine A; Waits, Lisette P; Finegan, Bryan

2017-09-01

Agricultural intensification in tropical landscapes poses a new threat to the ability of biological corridors to maintain functional connectivity for native species. We use a landscape genetics approach to evaluate impacts of expanding pineapple plantations on two widespread and abundant frugivorous bats in a biological corridor in Costa Rica. We hypothesize that the larger, more mobile Artibeus jamaicensis will be less impacted by pineapple than the smaller Carollia castanea. In 2012 and 2013, we sampled 735 bats in 26 remnant forest patches surrounded by different proportions of forest, pasture, crops and pineapple. We used 10 microsatellite loci for A. jamaicensis and 16 microsatellite loci for C. castanea to estimate genetic diversity and gene flow. Canonical correspondence analyses indicate that land cover type surrounding patches has no impact on genetic diversity of A. jamaicensis. However, for C. castanea, both percentage forest and pineapple surrounding patches explained a significant proportion of the variation in genetic diversity. Least-cost transect analyses (LCTA) and pairwise G″st suggest that for A. jamaicensis, pineapple is more permeable to gene flow than expected, while as expected, forest is the most permeable land cover for gene flow of C. castanea. For both species, LCTA indicate that development may play a role in inhibiting gene flow. The current study answers the call for landscape genetic research focused on tropical and agricultural landscapes, highlights the value of comparative landscape genetics in biological corridor design and management and is one of the few studies of biological corridors in any ecosystem to implement a genetic approach to test corridor efficacy. © 2017 John Wiley & Sons Ltd.

Quality changes and nutrient retention in fresh-cut versus whole fruits during storage.

PubMed

Gil, María I; Aguayo, Encarna; Kader, Adel A

2006-06-14

The influences of processing and storage on the quality indices and nutritional content of fresh-cut fruits were evaluated in comparison to whole fruits stored for the same duration but prepared on the day of sampling. Fresh-cut pineapples, mangoes, cantaloupes, watermelons, strawberries, and kiwifruits and whole fruits were stored for up to 9 days in air at 5 degrees C. The postcutting life based on visual appearance was shorter than 6 days for fresh-cut kiwifruit and shorter than 9 days for fresh-cut pineapple, cantaloupe, and strawberry. On the other hand, fresh-cut watermelon and mango pieces were still marketable after 9 days at 5 degrees C. Losses in vitamin C after 6 days at 5 degrees C were < or = 5% in mango, strawberry, and watermelon pieces, 10% in pineapple pieces, 12% in kiwifruit slices, and 25% in cantaloupe cubes. No losses in carotenoids were found in kiwifruit slices and watermelon cubes, whereas losses in pineapples were the highest at 25% followed by 10-15% in cantaloupe, mango, and strawberry pieces after 6 days at 5 degrees C. No significant losses in total phenolics were found in any of the fresh-cut fruit products tested after 6 days at 5 degrees C. Light exposure promoted browning in pineapple pieces and decreased vitamin C content in kiwifruit slices. Total carotenoids contents decreased in cantaloupe cubes and kiwifruit slices, but increased in mango and watermelon cubes in response to light exposure during storage at 5 degrees C for up to 9 days. There was no effect of exposure to light on the content of phenolics. In general, fresh-cut fruits visually spoil before any significant nutrient loss occurs.

Investigation of uterotonic properties of Ananas comosus extracts.

PubMed

Monji, Faezeh; Adaikan, P Ganesan; Lau, Lang Chu; Bin Said, Baharudin; Gong, Yinhan; Tan, Huey Min; Choolani, Mahesh

2016-12-04

In folklore medicine Ananas comosus (pineapple) is reputed to act as an abortifacient and in expectant women as a means of inducing labor. Several reports have claimed abortifacient property of A. comosus fruit (ripe or unripe). Ripe fruit has been used orally as traditional medicine in inducing abortion in Kerala state of India while the juice of unripe fruit was used for abortion in Bangladesh. However, scientific evidence supporting the efficacy of pineapple extracts in inducing uterine contractions is clearly lacking. This study investigated the pharmacological effects of different fractions of pineapple extract with a range of maturities to identify the most potent uterotonic fraction. The ethanolic crude extracts of pineapple (edible part) were prepared and fractionated through a series of liquid-liquid partitions. Fractions were separately tested on isolated uterine muscle from pregnant SD rats and human pregnant myometrium, which were cut into strips along the longitudinal axis of uterus. The strips were mounted vertically in organ baths (37°C) and exposed to cumulative addition of fractions (0.1-10mgml -1 ), serotonin (0.05-5µM) and different inhibitors to delineate the mechanism of action of the active ingredients of the extract. Aqueous fraction (F4) possesses uterine stimulant property which was blocked by verapamil but unaffected by indomethacin, prazosin and atosiban. Notably, ketanserin (10µM) diminished the maximal contractile response induced by both F4 and 5HT by 74.3% and 92.1% respectively. These results may indicate the presence of 5HT or 5HT-like compound(s) and serotonergic pathways may contribute to the uterotonic activity of pineapple extract. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Genomic Survey, Characterization, and Expression Profile Analysis of the SBP Genes in Pineapple (Ananas comosus L.).

PubMed

Ali, Hina; Liu, Yanhui; Azam, Syed Muhammad; Rahman, Zia Ur; Priyadarshani, S V G N; Li, Weimin; Huang, Xinyu; Hu, Bingyan; Xiong, Junjie; Ali, Umair; Qin, Yuan

2017-01-01

Gene expression is regulated by transcription factors, which play many significant developmental processes. SQUAMOSA promoter-binding proteins (SBP) perform a variety of regulatory functions in leaf, flower, and fruit development, plant architecture, and sporogenesis. 16 SBP genes were identified in pineapple and were divided into four groups on basis of phylogenetic analysis. Five paralogs in pineapple for SBP genes were identified with Ka/Ks ratio varied from 0.20 for AcSBP14 and AcSBP15 to 0.36 for AcSBP6 and AcSBP16 , respectively. 16 SBP genes were located on 12 chromosomes out of 25 pineapple chromosomes with highly conserved protein sequence structures. The isoionic points of SBP ranged from 6.05 to 9.57, while molecular weight varied from 22.7 to 121.9 kD. Expression profiles of SBP genes revealed that AcSBP7 and AcSBP15 (leaf), AcSBP13 , AcSBP12 , AcSBP8 , AcSBP16 , AcSBP9 , and AcSBP11 (sepal), AcSBP6 , AcSBP4 , and AcSBP10 (stamen), AcSBP14 , AcSBP1 , and AcSBP5 (fruit) while the rest of genes showed low expression in studied tissues. Four genes, that is, AcSBP11 , AcSBP6 , AcSBP4 , and AcSBP12 , were highly expressed at 4°C, while AcSBP16 were upregulated at 45°C. RNA-Seq was validated through qRT-PCR for some genes. Salt stress-induced expression of two genes, that is, AcSBP7 and AcSBP14 , while in drought stress, AcSBP12 and AcSBP15 were highly expressed. Our study lays a foundation for further gene function and expression studies of SBP genes in pineapple.

Genomic Survey, Characterization, and Expression Profile Analysis of the SBP Genes in Pineapple (Ananas comosus L.)

PubMed Central

Ali, Hina; Liu, Yanhui; Azam, Syed Muhammad; Rahman, Zia ur; Priyadarshani, S. V. G. N.; Li, Weimin; Huang, Xinyu; Hu, Bingyan; Xiong, Junjie; Ali, Umair

2017-01-01

Gene expression is regulated by transcription factors, which play many significant developmental processes. SQUAMOSA promoter-binding proteins (SBP) perform a variety of regulatory functions in leaf, flower, and fruit development, plant architecture, and sporogenesis. 16 SBP genes were identified in pineapple and were divided into four groups on basis of phylogenetic analysis. Five paralogs in pineapple for SBP genes were identified with Ka/Ks ratio varied from 0.20 for AcSBP14 and AcSBP15 to 0.36 for AcSBP6 and AcSBP16, respectively. 16 SBP genes were located on 12 chromosomes out of 25 pineapple chromosomes with highly conserved protein sequence structures. The isoionic points of SBP ranged from 6.05 to 9.57, while molecular weight varied from 22.7 to 121.9 kD. Expression profiles of SBP genes revealed that AcSBP7 and AcSBP15 (leaf), AcSBP13, AcSBP12, AcSBP8, AcSBP16, AcSBP9, and AcSBP11 (sepal), AcSBP6, AcSBP4, and AcSBP10 (stamen), AcSBP14, AcSBP1, and AcSBP5 (fruit) while the rest of genes showed low expression in studied tissues. Four genes, that is, AcSBP11, AcSBP6, AcSBP4, and AcSBP12, were highly expressed at 4°C, while AcSBP16 were upregulated at 45°C. RNA-Seq was validated through qRT-PCR for some genes. Salt stress-induced expression of two genes, that is, AcSBP7 and AcSBP14, while in drought stress, AcSBP12 and AcSBP15 were highly expressed. Our study lays a foundation for further gene function and expression studies of SBP genes in pineapple. PMID:29104869

Comparison of in vivo toxicity, antioxidant and immunomodulatory activities of coconut, nipah and pineapple juice vinegars.

PubMed

Mohamad, Nurul Elyani; Keong Yeap, Swee; Beh, Boon Keen; Romli, Muhammad Firdaus; Yusof, Hamidah Mohd; Kristeen-Teo, Ye Wen; Sharifuddin, Shaiful Adzni; Long, Kamariah; Alitheen, Noorjahan Banu

2018-01-01

Vinegar is widely used as a food additive, in food preparation and as a food supplement. This study compared the phenolic acid profiles and in vivo toxicities, and antioxidant and immunomodulatory effects of coconut, nipah and pineapple juice vinegars, which were respectively prepared via a two-step fermentation using Saccharomyces cerevisiae 7013 INRA and Acetobacter aceti vat Europeans. Pineapple juice vinegar, which had the highest total phenolic acid content, also exhibited the greatest in vitro antioxidant capacity compared to coconut juice and nipah juice vinegars. Following acute and sub-chronic in vivo toxicity evaluation, no toxicity and mortality were evident and there were no significant differences in the serum biochemical profiles between mice administered the vinegars versus the control group. In the sub-chronic toxicity evaluation, the highest liver antioxidant levels were found in mice fed with pineapple juice vinegar, followed by coconut juice and nipah juice vinegars. However, compared to the pineapple juice and nipah juice vinegars, the mice fed with coconut juice vinegar, exhibited a higher population of CD4 + and CD8 + T-lymphocytes in the spleen, which was associated with greater levels of serum interleukin-2 and interferon-γ cytokines. Overall, the data suggested that not all vinegar samples cause acute and sub-chronic toxicity in vivo. Moreover, the in vivo immunity and organ antioxidant levels were enhanced, to varying extents, by the phenolic acids present in the vinegars. The results obtained in this study provide appropriate guidelines for further in vivo bioactivity studies and pre-clinical assessments of vinegar consumption. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Cloning and Functional Characterization of a Gene for Capsanthin-Capsorubin Synthase from Tiger Lily (Lilium lancifolium Thunb. ‘Splendens’)

PubMed Central

Chen, Tony H.H.

2012-01-01

The orange color of tiger lily (Lolium lancifolium ‘Splendens’) flowers is due, primarily, to the accumulation of two κ-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene β-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription–PCR (RT–PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native κ-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of κ-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species. PMID:23008421

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orfH522 induces male sterility in transgenic tobacco plants.

PubMed

Nizampatnam, Narasimha Rao; Doodhi, Harinath; Kalinati Narasimhan, Yamini; Mulpuri, Sujatha; Viswanathaswamy, Dinesh Kumar

2009-03-01

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility.

Molecular cloning and functional analysis of the phosphomannomutase (PMM) gene from Dendrobium officinale and evidence for the involvement of an abiotic stress response during germination.

PubMed

He, Chunmei; Zeng, Songjun; Teixeira da Silva, Jaime A; Yu, Zhenming; Tan, Jianwen; Duan, Jun

2017-07-01

Phosphomannomutase (PMM, EC 5.4.2.8) catalyzes the interconversion of mannose-6-phosphate to mannose-1-phosphate, the precursor for the synthesis of GDP-mannose. In this study, the complementary DNA (cDNA) of the Phosphomannomutase (PMM) gene was initially cloned from Dendrobium officinale by RACE method. Transient transform result showed that the DoPMM protein was localized in the cytoplasm. The DoPMM gene was highly expressed in the stems of D. officinale both in vegetative and reproductive developmental stages. The putative promoter was cloned by TAIL-PCR and used for searched cis-elements. Stress-related cis-elements like ABRE, TCA-element, and MBS were found in the promoter regions. The DoPMM gene was up-regulated after treatment with abscisic acid, salicylic acid, cold, polyethylene glycol, and NaCl. The total ascorbic acid (AsA) and polysaccharide content in all of the 35S::DoPMM Arabidopsis thaliana transgenic lines #1, #2, and #5 showed a 40, 39, and 31% increase in AsA and a 77, 22, and 39% increase in polysaccharides, respectively more than wild-type (WT) levels. All three 35S::DoPMM transgenic lines exhibited a higher germination percentage than WT plants when seeded on half-strength MS medium supplemented with 150 mM NaCl or 300 mM mannitol. These results provide genetic evidence for the involvement of PMM genes in the biosynthesis of AsA and polysaccharides and the mediation of PMM genes in abiotic stress tolerance during seed germination in A. thaliana.

An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van De Loo, F.J.; Broun, P.; Turner, S.

1995-07-18

Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 andmore » with {approx}67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution. 26 refs., 6 figs., 1 tab.« less

Universality of clone dynamics during tissue development

NASA Astrophysics Data System (ADS)

Rulands, Steffen; Lescroart, Fabienne; Chabab, Samira; Hindley, Christopher J.; Prior, Nicole; Sznurkowska, Magdalena K.; Huch, Meritxell; Philpott, Anna; Blanpain, Cedric; Simons, Benjamin D.

2018-05-01

The emergence of complex organs is driven by the coordinated proliferation, migration and differentiation of precursor cells. The fate behaviour of these cells is reflected in the time evolution of their progeny, termed clones, which serve as a key experimental observable. In adult tissues, where cell dynamics is constrained by the condition of homeostasis, clonal tracing studies based on transgenic animal models have advanced our understanding of cell fate behaviour and its dysregulation in disease1,2. But what can be learnt from clonal dynamics in development, where the spatial cohesiveness of clones is impaired by tissue deformations during tissue growth? Drawing on the results of clonal tracing studies, we show that, despite the complexity of organ development, clonal dynamics may converge to a critical state characterized by universal scaling behaviour of clone sizes. By mapping clonal dynamics onto a generalization of the classical theory of aerosols, we elucidate the origin and range of scaling behaviours and show how the identification of universal scaling dependences may allow lineage-specific information to be distilled from experiments. Our study shows the emergence of core concepts of statistical physics in an unexpected context, identifying cellular systems as a laboratory to study non-equilibrium statistical physics.

Chemical and antioxidant properties of snake tomato (Trichosanthes cucumerina) juice and Pineapple (Ananas comosus) juice blends and their changes during storage.

PubMed

Bamidele, Oluwaseun P; Fasogbon, Mofoluwaso B

2017-04-01

Juice blends made from the mixture of snake tomato (Trichosanthes cucumerina) and Pineapple (Ananas comosus) fruits were analyzed for pH, antioxidant properties, total titratable acidity, vitamin C, lycopene and total phenolic contents after different blend ratios were made. The addition of snake tomato juice increased the vitamin C, total carotene, lycopene and antioxidant properties of the juice blends. The radical scavenging properties of juice blends containing a higher ratio of snake tomato were higher and samples stored at room temperature (29°C) showed an increase in antioxidant properties compared to samples stored at 4°C. In conclusion, snake tomato juice up to 50% may be added to Pineapple juice to make a healthy juice blend. Copyright © 2016 Elsevier Ltd. All rights reserved.

Community Geothermal Technology Program: Fruit drying with geothermal energy. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1988-03-14

Largest problem was lack of proper recording and controlling instrumentation. Agricultural products tested were green papaya powder, banana slices, and pineapple slices. Results show that a temperature of 120 F is a good drying temperature. Papaya should be mature green and not overly ripe; banana ripeness is also important; and pineapple slice thickness should be very uniform for even drying. Geothermal drying is feasible. Figs, tabs.

Survival and expression of acid resistance genes in Shiga toxin-producing Escherichia coli acid adapted in pineapple juice and exposed to synthetic gastric fluid

USDA-ARS?s Scientific Manuscript database

Aims: The aim of this research was to examine relative transcriptional expression of acid resistance (AR) genes, rpoS, gadA and adiA, in O157:H7 and non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes after adaptation to pineapple juice (PJ) and subsequently to determine survival with e...

Biotransformation of pineapple juice sugars into dietetic derivatives by using a cell free oxidoreductase from Zymomonas mobilis together with commercial invertase.

PubMed

Aziz, M G; Michlmayr, H; Kulbe, K D; Del Hierro, A M

2011-01-05

An easy procedure for cell free biotransformation of pineapple juice sugars into dietetic derivatives was accomplished using a commercial invertase and an oxidoreductase from Zymomonas mobilis. First, pineapple juice sucrose was quantitatively converted into glucose and fructose by invertase, thus increasing the concentration of each monosaccharide in the original juice to almost twice. In a second step, glucose-fructose oxidoreductase (GFOR) transformed glucose into gluconolactone, and fructose into the low calorie sweetener sorbitol. The advantage of using GFOR is simultaneous reduction of fructose and oxidation of glucose, allowing the continuous regeneration of the essential coenzyme NADP(H), that is tightly bound to the enzyme. The yield of GFOR catalyzed sugar conversion depends on initial pH and control of pH during the reaction. At optimal conditions (pH control at 6.2) a maximum of 80% (w/v) sugar conversion was obtained. Without pH control, GFOR is inactivated rapidly due to gluconic acid formation. Therefore, conversion yields are relatively low at the natural pH of pineapple juice. The application of this process might be more advantageous on juices of other tropical fruits (papaya, jackfruit, mango) due to their naturally given higher pH. Copyright © 2010 Elsevier Inc. All rights reserved.

Fabrication, mechanical characterization of pineapple leaf fiber (PALF) reinforced vinylester hybrid composites

NASA Astrophysics Data System (ADS)

Yogesh, M.; Rao, A. N. Hari

2018-04-01

Natural fibre based composites are under intensive study due to their eco friendly nature and peculiar properties. The advantage of natural fibres is their continuous supply, easy and safe handling, and biodegradable nature. Although natural fibres exhibit admirable physical and mechanical properties, it varies with the plant source, species, geography, and so forth. Pineapple leave fibre (PALF) is one of the abundantly available waste materials in India and has not been studied yet. The work has been carried out to fabrication and study the mechanical characterization of Pineapple Leaf fiber reinforced Vinylester composites filled with different particulate fillers. These results are compared with those of a similar set of glass fiber reinforced Vinylester composites filled with same particulate fillers. It is evident that the density values for Pineapple leaf fiber (PALF) - Vinylester composites increase with the particulate filler content and void fractions in these composites also increase. The test results show that with the presence of particulate fillers, micro hardness of the PALF-Vinylester composites has improved. Among all the composites under this investigation, the maximum hardness value is recorded for PALF-Vinylester composite filled with 20 wt% alumina. In this investigation the maximum value of ILSS has been recorded for the PALF-Vinylester composite with 20 wt% of Flyash.

Mapping the rainfall distribution for irrigation planning in dry season at pineapple plantation, Lampung Province, Indonesia (Study case at Great Giant Pineapple Co. Ltd.)

NASA Astrophysics Data System (ADS)

Cahyono, P.; Astuti, N. K.; Purwito; Rahmat, A.

2018-03-01

One of the problems caused by climate change is unpredictable of the dry season. Understanding when the dry season will start is very important to planning the irrigation schedule especially on large plantation. Data of rainfall for 30 years in Lampung, especially in Pineapple Plantation show that dry month occurs from June to October. If in two decadals (ten days period) rainfall less than 100 mm then it is predicted that next decadal will be dry season. Great Giant Pineapple Co. Ltd. has 32,000 hectares plantation area and located in three regencies at Lampung Province, Indonesia with varies rainfall between regions within a plantation. Therefore, monitoring the rainfall distribution by using ombrometer installed at 10 representative location points can be used to determine irrigation period at the beginning of dry season. Mapping method using the server program and data source is from 10 monitoring rainfall stations installed at the observed points. Preparation of rainfall distribution mapping is important to know the beginning of the dry season and thus planning the irrigation. The results show that 2nd decadal of April is indicated as the starting time of dry season, which is similar with Indonesian government for climate agency’s result.

Glutathione Transferase from Trichoderma virens Enhances Cadmium Tolerance without Enhancing Its Accumulation in Transgenic Nicotiana tabacum

PubMed Central

Dixit, Prachy; Mukherjee, Prasun K.; Ramachandran, V.; Eapen, Susan

2011-01-01

Background Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. Results Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. Conclusion The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and in limiting Cd availability in the food chain. PMID:21283689

Conservative site-specific and single-copy transgenesis in human LINE-1 elements

PubMed Central

Vijaya Chandra, Shree Harsha; Makhija, Harshyaa; Peter, Sabrina; Myint Wai, Cho Mar; Li, Jinming; Zhu, Jindong; Ren, Zhonglu; D'Alcontres, Martina Stagno; Siau, Jia Wei; Chee, Sharon; Ghadessy, Farid John; Dröge, Peter

2016-01-01

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, termed attH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes. PMID:26673710

Co-Expression of Monodehydroascorbate Reductase and Dehydroascorbate Reductase from Brassica rapa Effectively Confers Tolerance to Freezing-Induced Oxidative Stress

PubMed Central

Shin, Sun-Young; Kim, Myung-Hee; Kim, Yul-Ho; Park, Hyang-Mi; Yoon, Ho-Sung

2013-01-01

Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing. PMID:24170089

JPRS Report, Science and Technology USSR: Life Sciences.

DTIC Science & Technology

1990-09-26

newly observed smut flowers infected with Ustilago vaillantii smut, infection parasite does not destroy the host, so it probably does of its spores by...designed to further expand studies on transgenic plants. selected on hygromycin B. All characterized clones syn- pABDI was constructed from Escherichia coli...they numbered more than 2,000 in July- farms into four groups, according to number of workers: September. In livestock breeding , the largest number of

Cloning of Gossypium hirsutum Sucrose Non-Fermenting 1-Related Protein Kinase 2 Gene (GhSnRK2) and Its Overexpression in Transgenic Arabidopsis Escalates Drought and Low Temperature Tolerance

PubMed Central

Bello, Babatunde; Zhang, Xueyan; Liu, Chuanliang; Yang, Zhaoen; Yang, Zuoren; Wang, Qianhua; Zhao, Ge; Li, Fuguang

2014-01-01

The molecular mechanisms of stress tolerance and the use of modern genetics approaches for the improvement of drought stress tolerance have been major focuses of plant molecular biologists. In the present study, we cloned the Gossypium hirsutum sucrose non-fermenting 1-related protein kinase 2 (GhSnRK2) gene and investigated its functions in transgenic Arabidopsis. We further elucidated the function of this gene in transgenic cotton using virus-induced gene silencing (VIGS) techniques. We hypothesized that GhSnRK2 participates in the stress signaling pathway and elucidated its role in enhancing stress tolerance in plants via various stress-related pathways and stress-responsive genes. We determined that the subcellular localization of the GhSnRK2-green fluorescent protein (GFP) was localized in the nuclei and cytoplasm. In contrast to wild-type plants, transgenic plants overexpressing GhSnRK2 exhibited increased tolerance to drought, cold, abscisic acid and salt stresses, suggesting that GhSnRK2 acts as a positive regulator in response to cold and drought stresses. Plants overexpressing GhSnRK2 displayed evidence of reduced water loss, turgor regulation, elevated relative water content, biomass, and proline accumulation. qRT-PCR analysis of GhSnRK2 expression suggested that this gene may function in diverse tissues. Under normal and stress conditions, the expression levels of stress-inducible genes, such as AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1, and AtABI5, were increased in the GhSnRK2-overexpressing plants compared to the wild-type plants. GhSnRK2 gene silencing alleviated drought tolerance in cotton plants, indicating that VIGS technique can certainly be used as an effective means to examine gene function by knocking down the expression of distinctly expressed genes. The results of this study suggested that the GhSnRK2 gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance. PMID:25393623

Antisense expression of the fasciclin-like arabinogalactan protein FLA6 gene in Populus inhibits expression of its homologous genes and alters stem biomechanics and cell wall composition in transgenic trees

PubMed Central

Wang, Haihai; Jiang, Chunmei; Wang, Cuiting; Yang, Yang; Yang, Lei; Gao, Xiaoyan; Zhang, Hongxia

2015-01-01

Fasciclin-like arabinogalactan proteins (FLAs) play important roles in the growth and development of roots, stems, and seeds in Arabidopsis. However, their biological functions in woody plants are largely unknown. In this work, we investigated the possible function of PtFLA6 in poplar. Quantitative real-time PCR, PtFLA6–yellow fluorescent protein (YFP) fusion protein subcellular localization, Western blotting, and immunohistochemical analyses demonstrated that the PtFLA6 gene was expressed specifically in the xylem of mature stem, and PtFLA6 protein was distributed ubiquitous in plant cells and accumulated predominantly in stem xylem fibres. Antisense expression of PtFLA6 in the aspen hybrid clone Poplar davidiana×Poplar bolleana reduced the transcripts of PtFLA6 and its homologous genes. Transgenic plants that showed a significant reduction in the transcripts of PtFLAs accumulated fewer PtFLA6 and arabinogalactan proteins than did the non-transgenic plants, leading to reduced stem flexural strength and stiffness. Further studies revealed that the altered stem biomechanics of transgenic plants could be attributed to the decreased cellulose and lignin composition in the xylem. In addition expression of some xylem-specific genes involved in cell wall biosynthesis was downregulated in these transgenic plants. All these results suggest that engineering the expression of PtFLA6 and its homologues could modulate stem mechanical properties by affecting cell wall composition in trees. PMID:25428999

DoGMP1 from Dendrobium officinale contributes to mannose content of water-soluble polysaccharides and plays a role in salt stress response

PubMed Central

He, Chunmei; Yu, Zhenming; Teixeira da Silva, Jaime A.; Zhang, Jianxia; Liu, Xuncheng; Wang, Xiaojuan; Zhang, Xinhua; Zeng, Songjun; Wu, Kunlin; Tan, Jianwen; Ma, Guohua; Luo, Jianping; Duan, Jun

2017-01-01

GDP-mannose pyrophosphorylase (GMP) catalyzed the formation of GDP-mannose, which serves as a donor for the biosynthesis of mannose-containing polysaccharides. In this study, three GMP genes from Dendrobium officinale (i.e., DoGMPs) were cloned and analyzed. The putative 1000 bp upstream regulatory region of these DoGMPs was isolated and cis-elements were identified, which indicates their possible role in responses to abiotic stresses. The DoGMP1 protein was shown to be localized in the cytoplasm. To further study the function of the DoGMP1 gene, 35S:DoGMP1 transgenic A. thaliana plants with an enhanced expression level of DoGMP1 were generated. Transgenic plants were indistinguishable from wild-type (WT) plants in tissue culture or in soil. However, the mannose content of the extracted water-soluble polysaccharides increased 67%, 96% and 92% in transgenic lines #1, #2 and #3, respectively more than WT levels. Germination percentage of seeds from transgenic lines was higher than WT seeds and the growth of seedlings from transgenic lines was better than WT seedlings under salinity stress (150 mM NaCl). Our results provide genetic evidence for the involvement of GMP genes in the biosynthesis of mannose-containing polysaccharides and the mediation of GMP genes in the response to salt stress during seed germination and seedling growth. PMID:28176760

Molecular genetic transfection of the coccidian parasite Sarcocystis neurona.

PubMed

Gaji, Rajshekhar Y; Zhang, Deqing; Breathnach, Cormac C; Vaishnava, Shipra; Striepen, Boris; Howe, Daniel K

2006-11-01

Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

A Thermostable β-Glucuronidase Obtained by Directed Evolution as a Reporter Gene in Transgenic Plants

PubMed Central

Xiong, Ai-Sheng; Peng, Ri-He; Zhuang, Jing; Chen, Jian-Min; Zhang, Bin; Zhang, Jian; Yao, Quan-Hong

2011-01-01

A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter. PMID:22096498

Fingerprinting and quantification of GMOs in the agro-food sector.

PubMed

Taverniers, I; Van Bockstaele, E; De Loose, M

2003-01-01

Most strategies for analyzing GMOs in plants and derived food and feed products, are based on the polymerase chain reaction (PCR) technique. In conventional PCR methods, a 'known' sequence between two specific primers is amplified. To the contrary, with the 'anchor PCR' technique, unknown sequences adjacent to a known sequence, can be amplified. Because T-DNA/plant border sequences are being amplified, anchor PCR is the perfect tool for unique identification of transgenes, including non-authorized GMOs. In this work, anchor PCR was applied to characterize the 'transgene locus' and to clarify the complete molecular structure of at least six different commercial transgenic plants. Based on sequences of T-DNA/plant border junctions, obtained by anchor PCR, event specific primers were developed. The junction fragments, together with endogeneous reference gene targets, were cloned in plasmids. The latter were then used as event specific calibrators in real-time PCR, a new technique for the accurate relative quantification of GMOs. We demonstrate here the importance of anchor PCR for identification and the usefulness of plasmid DNA calibrators in quantification strategies for GMOs, throughout the agro-food sector.

Epithelial reticulon 4B (Nogo-B) is an endogenous regulator of Th2-driven lung inflammation

PubMed Central

Wright, Paulette L.; Yu, Jun; Di, Y.P. Peter; Homer, Robert J.; Chupp, Geoffrey; Elias, Jack A.; Cohn, Lauren

2010-01-01

Nogo-B is a member of the reticulon family of proteins (RTN-4B) that is highly expressed in lung tissue; however, its function remains unknown. We show that mice with Th2-driven lung inflammation results in a loss of Nogo expression in airway epithelium and smooth muscle compared with nonallergic mice, a finding which is replicated in severe human asthma. Mice lacking Nogo-A/B (Nogo-KO) display an exaggerated asthma-like phenotype, and epithelial reconstitution of Nogo-B in transgenic mice blunts Th2-mediated lung inflammation. Microarray analysis of lungs from Nogo-KO mice reveals a marked reduction in palate lung and nasal clone (PLUNC) gene expression, and the levels of PLUNC are enhanced in epithelial Nogo-B transgenic mice. Finally, transgenic expression of PLUNC into Nogo-KO mice rescues the enhanced asthmatic-like responsiveness in these KO mice. These data identify Nogo-B as a novel protective gene expressed in lung epithelia, and its expression regulates the levels of the antibacterial antiinflammatory protein PLUNC. PMID:20975041

16S Ribosomal DNA Characterization of Nitrogen-Fixing Bacteria Isolated from Banana (Musa spp.) and Pineapple (Ananas comosus (L.) Merril)

PubMed Central

Magalhães Cruz, Leonardo; Maltempi de Souza, Emanuel; Weber, Olmar Baler; Baldani, José Ivo; Döbereiner, Johanna; de Oliveira Pedrosa, Fábio

2001-01-01

Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria. PMID:11319127

The pineapple AcMADS1 promoter confers high level expression in tomato and arabidopsis flowering and fruiting tissues, but AcMADS1 does not complement the tomato LeMADS-RIN (rin) mutant

USDA-ARS?s Scientific Manuscript database

A previous EST study identified a MADS box transcription factor coding sequence, AcMADS1, that is strongly induced during non-climacteric pineapple fruit ripening. Phylogenetic analyses place the AcMADS1 protein in the same superclade as LeMADS-RIN, a master regulator of fruit ripening upstream of e...

Research of Uncertainty Reasoning in Pineapple Disease Identification System

NASA Astrophysics Data System (ADS)

Liu, Liqun; Fan, Haifeng

In order to deal with the uncertainty of evidences mostly existing in pineapple disease identification system, a reasoning model based on evidence credibility factor was established. The uncertainty reasoning method is discussed,including: uncertain representation of knowledge, uncertain representation of rules, uncertain representation of multi-evidences and update of reasoning rules. The reasoning can fully reflect the uncertainty in disease identification and reduce the influence of subjective factors on the accuracy of the system.

Reverse micellar extraction of bromelain from pineapple peel--Effect of surfactant structure.

PubMed

Wan, Jing; Guo, Jingjing; Miao, Zhitong; Guo, Xia

2016-04-15

Pineapple peel is generally disposed or used as compost. This study was focused on extracting bromelain from pineapple peel by using reverse micelles. It was found that gemini surfactant C12-8-C12·2Br (octamethylene-α,ω-bis(dimethyldodecylammonium bromide)) showed distinctive advantage over its monomeric counterpart DTAB (dodecyl trimethyl ammonium bromide); under optimized condition, the bromelain extracted with C12-8-C12·2Br reverse micelle had an activity recovery of 163% and a purification fold of 3.3, while when using DTAB reverse micelle, the activity recovery was 95% and the purification fold was 1.7. Therefore, the spacer of gemini surfactant should play a positive role in bromelain extraction and may suggest the potential of gemini surfactant in protein separation since it has been so far rarely used in relative experiments or technologies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Pollen-mediated gene flow from transgenic to non-transgenic switchgrass (Panicum virgatum L.) in the field.

PubMed

Millwood, Reginald; Nageswara-Rao, Madhugiri; Ye, Rongjian; Terry-Emert, Ellie; Johnson, Chelsea R; Hanson, Micaha; Burris, Jason N; Kwit, Charles; Stewart, C Neal

2017-05-02

Switchgrass is C 4 perennial grass species that is being developed as a cellulosic bioenergy feedstock. It is wind-pollinated and considered to be an obligate outcrosser. Genetic engineering has been used to alter cell walls for more facile bioprocessing and biofuel yield. Gene flow from transgenic cultivars would likely be of regulatory concern. In this study we investigated pollen-mediated gene flow from transgenic to nontransgenic switchgrass in a 3-year field experiment performed in Oliver Springs, Tennessee, U.S.A. using a modified Nelder wheel design. The planted area (0.6 ha) contained sexually compatible pollen source and pollen receptor switchgrass plants. One hundred clonal switchgrass 'Alamo' plants transgenic for an orange-fluorescent protein (OFP) and hygromycin resistance were used as the pollen source; whole plants, including pollen, were orange-fluorescent. To assess pollen movement, pollen traps were placed at 10 m intervals from the pollen-source plot in the four cardinal directions extending to 20 m, 30 m, 30 m, and 100 m to the north, south, west, and east, respectively. To assess pollination rates, nontransgenic 'Alamo 2' switchgrass clones were planted in pairs adjacent to pollen traps. In the eastward direction there was a 98% decrease in OFP pollen grains from 10 to 100 m from the pollen-source plot (Poisson regression, F1,8 = 288.38, P < 0.0001). At the end of the second and third year, 1,820 F 1 seeds were collected from pollen recipient-plots of which 962 (52.9%) germinated and analyzed for their transgenic status. Transgenic progeny production detected in each pollen-recipient plot decreased with increased distance from the edge of the transgenic plot (Poisson regression, F1,15 = 12.98, P < 0.003). The frequency of transgenic progeny detected in the eastward plots (the direction of the prevailing wind) ranged from 79.2% at 10 m to 9.3% at 100 m. In these experiments we found transgenic pollen movement and hybridization rates to be inversely associated with distance. However, these data suggest pollen-mediated gene flow is likely to occur up to, at least, 100 m. This study gives baseline data useful to determine isolation distances and other management practices should transgenic switchgrass be grown commercially in relevant environments.

Overexpression of STARCH BRANCHING ENZYME II increases short-chain branching of amylopectin and alters the physicochemical properties of starch from potato tuber.

PubMed

Brummell, David A; Watson, Lyn M; Zhou, Jun; McKenzie, Marian J; Hallett, Ian C; Simmons, Lyall; Carpenter, Margaret; Timmerman-Vaughan, Gail M

2015-04-29

Starch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers. A hybrid SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6-12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern. This work reports the first time that overexpression of SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.

An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin-enriched milk in sheep.

PubMed

Ma, Teng; Tao, Jingli; Yang, Minghui; He, Changjiu; Tian, Xiuzhi; Zhang, Xiaosheng; Zhang, Jinlong; Deng, Shoulong; Feng, Jianzhong; Zhang, Zhenzhen; Wang, Jing; Ji, Pengyun; Song, Yukun; He, Pingli; Han, Hongbing; Fu, Juncai; Lian, Zhengxing; Liu, Guoshi

2017-08-01

Melatonin as a potent antioxidant exhibits important nutritional and medicinal values. To produce melatonin-enriched milk will benefit the consumers. In this study, a sheep bioreactor which generates melatonin-enriched milk has been successfully developed by the technology that combined CRISPR/Cas9 system and microinjection. The AANAT and ASMT were cloned from pineal gland of Dorper sheep (Ovis aries). The in vitro studies found that AANAT and ASMT were successfully transferred to the mammary epithelial cell lines and significantly increased melatonin production in the culture medium compared to the nontransgenic cell lines. In addition, the Cas9 mRNA, sgRNA, and the linearized vectors pBC1-AANAT and pBC1-ASMT were co-injected into the cytoplasm of pronuclear embryos which were implanted into ewes by oviducts transferring. Thirty-four transgenic sheep were generated with the transgenic positive rate being roughly 35% which were identified by Southern blot and sequencing. Seven carried transgenic AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. RT-PCR and Western blot demonstrated that the lambs expressed these genes in their mammary epithelial cells and these animals produced melatonin-enriched milk. This is the first report to show a functional AANAT and ASMT transgenic animal model which produce significantly high levels of melatonin milk compared to their wild-type counterparts. The advanced technologies used in the study laid a foundation for generating large transgenic livestock, for example, the cows, which can produce high level of melatonin milk. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

No impact of transgenic nptII-leafy Pinus radiata (Pinales: Pinaceae) on Pseudocoremia suavis (Lepidoptera: Geometridae) or its endoparasitoid Meteorus pulchricornis (Hymenoptera: Braconidae).

PubMed

Burgess, E P J; Barraclough, E I; Kean, A M; Walter, C; Malone, L A

2011-10-01

To investigate the biosafety to insects of transgenic Pinus radiata D. Don containing the antibiotic resistance marker gene nptII and the reproductive control gene leafy, bioassays were conducted with an endemic lepidopteran pest of New Zealand plantation pine forests and a hymenopteran endoparasitoid. Larvae of the common forest looper, Pseudocoremia suavis (Butler), were fed from hatching on P. radiata needles from either one of two nptII-leafy transgenic clones, or an isogenic unmodified control line. For both unparasitized P. suavis and those parasitized by Meteorus pulchricornis (Wesmael), consuming transgenic versus control pine had no impact on larval growth rate or mass at any age, larval duration, survival, pupation or successful emergence as an adult. Total larval duration was 1 d (3%) longer in larvae fed nptII-2 than nptII-1, but this difference was considered trivial and neither differed from the control. In unparasitized P. suavis larvae, pine type consumed did not affect rate of pupation or adult emergence, pupal mass, or pupal duration. Pine type had no effect on the duration or survival of M. pulchricornis larval or pupal stages, mass of cocoons, stage at which they died, adult emergence, or fecundity. Parasitism by M. pulchricornis reduced P. suavis larval growth rate, increased the duration of the third larval stadium, and resulted in the death of all host larvae before pupation. The lack of impact of an exclusive diet of nptII-leafy transgenic pines on the life history of P. suavis and M. pulchricornis suggests that transgenic plantation pines expressing nptII are unlikely to affect insect populations in the field.

Watermelon (Citrullus lanatus) hydroperoxide lyase greatly increases C6 aldehyde formation in transgenic leaves.

PubMed

Fukushige, Hirotada; Hildebrand, David F

2005-03-23

Fatty acid hydroperoxide lyase (HL) is the key enzyme for the production of the "green note"compounds, leaf aldehyde [(2E)-hexenal] and leaf alcohol [(3Z)-hexenol], in plant tissues. A cDNA encoding HL was cloned from leaves of watermelon (Citrullus lanatus) and expressed in Nicotiana tabacum. The enzyme is 3 times more active with 13-hydroperoxylinolenic acid than with 13-hydroperoxylinoleic acid. The activity against 9-hydroperoxides of polyunsaturated fatty acids is minimal. Enzyme activity of the watermelon HL in the transgenic leaves was approximately 50 times higher than endogenous HL activity in the wild-type N. tabacum plants. When compared with Arabidopsis HL also expressed in N. tabacum, the highest HL activity is 10 times higher in watermelon HL overexpressing leaves than in Arabidopsis HL overexpressers.

Role of pectolytic enzymes in the programmed separation of cells from the root cap of higher plants. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hawes, M.C.

1995-03-01

The objective of this research was to develop a model system to study border cell separation in transgenic pea roots. In addition, the hypothesis that genes encoding pectolytic enzymes in the root cap play a role in the programmed separation of root border cells from the root tip was tested. The following objectives have been accomplished: (1) the use of transgenic hairy roots to study border cell separation has been optimized for Pisum sativum; (2) a cDNA encoding a root cap pectinmethylesterase (PME) has been cloned; (3) PME and polygalacturonase activities in cell walls of the root cap have beenmore » characterized and shown to be correlated with border cell separation. A fusion gene encoding pectate lyase has also been transformed into pea hairy root cells.« less

Recombinant protein production from stable mammalian cell lines and pools.

PubMed

Hacker, David L; Balasubramanian, Sowmya

2016-06-01

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

In Vivo Imaging of mdrla Gene Expression

DTIC Science & Technology

2005-06-01

svImJ mouse strain, compatible with the ES cells used in our Transgenic Mouse Facility. b. Engineer PGK-neo and Renilla luciferase cassettes...inserted between the two loxP sites, upstream of the Neo cassette. A cloning strategy was then devised to fuse Renilla luciferase in-frame with the...sites: B, BamHI; E, EcoRI; S, ScaI. PGK-neo: neo under the control of the PGK promoter. Luc: Renilla luciferase fused in- frame with the translated

A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

PubMed Central

Lassner, M W; Lardizabal, K; Metz, J G

1996-01-01

beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils. PMID:8742713

A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

PubMed

Lassner, M W; Lardizabal, K; Metz, J G

1996-02-01

beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils.

Complementary DNA cloning of the pear 1-aminocyclopropane-1-carboxylic acid oxidase gene and agrobacterium-mediated anti-sense genetic transformation.

PubMed

Qi, Jing; Dong, Zhen; Zhang, Yu-Xing

2015-12-01

The aim of the present study was to genetically modify plantlets of the Chinese yali pear to reduce their expression of ripening-associated 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and therefore increase the shelf-life of the fruit. Primers were designed with selectivity for the conserved regions of published ACO gene sequences, and yali complementary DNA (cDNA) cloning was performed by reverse transcription quantitative polymerase chain reaction (PCR). The obtained cDNA fragment contained 831 base pairs, encoding 276 amino acid residues, and shared no less than 94% nucleotide sequence identity with other published ACO genes. The cDNA fragment was inversely inserted into a pBI121 expression vector, between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator, in order to construct the anti‑sense expression vector of the ACO gene; it was transfected into cultured yali plants using Agrobacterium LBA4404. Four independent transgenic lines of pear plantlets were obtained and validated by PCR analysis. A Southern blot assay revealed that there were three transgenic lines containing a single copy of exogenous gene and one line with double copies. The present study provided germplasm resources for the cultivation of novel storage varieties of pears, therefore providing a reference for further applications of anti‑sense RNA technology in the genetic improvement of pears and other fruit.

Molecular Cloning and Characterization of Violaxanthin De-Epoxidase (CsVDE) in Cucumber

PubMed Central

Huang, Hongyu; Kong, Lingcui; Niu, Dandan; Sui, Xiaolei; Zhang, Zhenxian

2013-01-01

Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under high light stress. We have cloned a violaxanthin de-epoxidase gene (CsVDE) from cucumber. The amino acid sequence of CsVDE has high homology with VDEs in other plants. RT-PCR analysis and histochemical staining show that CsVDE is expressed in all green tissues in cucumber and Arabidopsis. Using GFP fusion protein and immunogold labeling methods, we show that CsVDE is mainly localized in chloroplasts in cucumber. Under high light stress, relative expression of CsVDE and the de-epoxidation ratio (A+Z)/(V+A+Z) is increased rapidly, and abundance of the gold particles was also increased. Furthermore, CsVDE is quickly induced by cold and drought stress, reaching maximum levels at the 2nd hour and the 9th day, respectively. The ratio of (A+Z)/(V+A+Z) and non-photochemical quenching (NPQ) is reduced in transgenic Arabidopsis down-regulated by the antisense fragment of CsVDE, compared to wild type (WT) Arabidopsis under high light stress. This indicates decreased functionality of the xanthophyll cycle and increased sensitivity to photoinhibition of photosystem II (PSII) in transgenic Arabidopsis under high light stress. PMID:23717606

Enhance beef cattle improvement by embryo biotechnologies.

PubMed

Wu, B; Zan, L

2012-10-01

Embryo biotechnology has become one of the prominent high businesses worldwide. This technology has evolved through three major changes, that is, traditional embryo transfer (in vivo embryo production by donor superovulation), in vitro embryo production by ovum pick up with in vitro fertilization and notably current cloning technique by somatic cell nuclear transfer and transgenic animal production. Embryo biotechnology has widely been used in dairy and beef cattle industry and commercial bovine embryo transfer has become a large international business. Currently, many developed biotechnologies during the period from early oocyte stage to pre-implantation embryos can be used to create new animal breeds and accelerate genetic progression. Based on recent advances in embryo biotechnologies and authors current studies, this review will focus on a description of the application of this technology to beef cattle improvement and discuss how to use this technology to accelerate beef cattle breeding and production. The main topics of this presentation include the following: (i) how to increase calf production numbers from gametes including sperm and oocyte; (ii) multiple ovulation and embryo transfer breeding schemes; (iii) in vitro fertilization and intracytoplasm sperm injection in bovine; (iv) pronuclear development and transgenic animals; (v) sex selection from sperm and embryos; (vi) cloning and androgenesis; (vii) blastocyst development and embryonic stem cells; (viii) preservation of beef cattle genetic resources; and (ix) conclusions. © 2011 Blackwell Verlag GmbH.

Cloning and characterization of the Cerasus humilis sucrose phosphate synthase gene (ChSPS1)

PubMed Central

Du, Junjie; Mu, Xiaopeng; Wang, Pengfei

2017-01-01

Sucrose is crucial to the growth and development of plants, and sucrose phosphate synthase (SPS) plays a key role in sucrose synthesis. To understand the genetic and molecular mechanisms of sucrose synthesis in Cerasus humilis, ChSPS1, a homologue of SPS, was cloned using RT-PCR. Sequence analysis showed that the open reading frame (ORF) sequence of ChSPS1 is 3174 bp in length, encoding a predicted protein of 1057 amino acids. The predicted protein showed a high degree of sequence identity with SPS homologues from other species. Real-time RT-PCR analysis showed that ChSPS1 mRNA was detected in all tissues and the transcription level was the highest in mature fruit. There is a significant positive correlation between expression of ChSPS1 and sucrose content. Prokaryotic expression of ChSPS1 indicated that ChSPS1 protein was expressed in E. coli and it had the SPS activity. Overexpression of ChSPS1 in tobacco led to upregulation of enzyme activity and increased sucrose contents in transgenic plants. Real-time RT-PCR analysis showed that the expression of ChSPS1 in transgenic tobacco was significantly higher than in wild type plants. These results suggested that ChSPS1 plays an important role in sucrose synthesis in Cerasus humilis. PMID:29036229

Microbial, Physicochemical, and Sensory Characteristics of Quality Grade 2 Beef Enhanced by Injection of Pineapple Concentrate and Honey

PubMed Central

Yoon, Ji Won; Lee, Da Gyeom; Lee, Hyun Jung; Choe, Juhui; Jung, Samooel; Jo, Cheorun

2017-01-01

This study investigated the effect of injecting pineapple concentrate and honey into low marbled beef in order to enhance its sensory qualities, particularly tenderness and flavor, without compromising its fresh appearance. Beef loin was injected with a solution of 6.0% pineapple concentrate, 2.5% honey, 0.5% monosodium L-glutamate, 0.5% phosphate, and 0.3% salt (w/w) to 120% (w/w) of initial meat weight and stored for 14 d. Non-injected beef loin served as a control. Total aerobic bacterial counts, surface meat color, shear force, reducing sugar content, and sensory evaluation of the beef were analyzed at 0.5, 7, and 14 d of storage. Injection did not affect the total aerobic bacterial counts or color of the beef. However, injection increased the stability of meat color, compared with that of the control, during storage. The shear force value was significantly lower in the injected beef than that in the control. The injected beef had a significantly higher reducing sugar content compared with that of the control. In sensory evaluation, tenderness, juiciness, flavor, and overall acceptance of the injected beef were significantly higher than those of the control at 0.5 d. In conclusion, injection of pineapple concentrate and honey can improve the sensory qualities of low marbled beef, during short storage periods, without changing the fresh appearance of the beef. PMID:28943761

Effect of refrigerated storage on probiotic viability and the production and stability of antimutagenic and antioxidant peptides in yogurt supplemented with pineapple peel.

PubMed

Sah, B N P; Vasiljevic, T; McKechnie, S; Donkor, O N

2015-09-01

Fruit by-products are good resources of carbohydrates, proteins, vitamins, and minerals, which may function as growth nutrients for probiotic bacteria. This research aimed at evaluating effects of pineapple peel powder addition on the viability and activity of Lactobacillus acidophilus (ATCC 4356), Lactobacillus casei (ATCC393), and Lactobacillus paracasei ssp. paracasei (ATCC BAA52) in yogurts throughout storage at 4°C for 28d. Plain and probiotic yogurts supplemented with or without pineapple peel powder or inulin were prepared. The probiotic counts in supplemented yogurts at 28d of storage ranged from 7.68 and 8.03 log cfu/g, one log cycle higher compared with nonsupplemented control yogurt. Degree of proteolysis in synbiotic yogurts was significantly higher than plain yogurts and increased substantially during storage. Crude water-soluble peptide extract of the probiotic yogurt with peel possessed stronger antimutagenic and antioxidant activities [evaluated measuring reducing power and scavenging capacity of 1,1-diphenyl-2-picrylhydrazyl; 2,2'-azino-bis(3-ethyl benzothiazoline-6-sulfonic acid), and hydroxyl radicals] than control and maintained during storage. Pineapple peel, a by-product of juice production, could be proposed as a prebiotic ingredient in the manufacture of yogurts with enhanced nutrition, and functionality. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Biogas production from pineapple core - A preliminary study

NASA Astrophysics Data System (ADS)

Jehan, O. S.; Sanusi, S. N. A.; Sukor, M. Z.; Noraini, M.; Buddin, M. M. H. S.; Hamid, K. H. K.

2017-09-01

Anaerobic digestion of pineapple waste was investigated by using pineapple core as the sole substrate. Pineapple core was chosen due to its high total sugar content thus, indicating high amount of fermentable sugar. As digestion process requires the involvement of microorganisms, wastewater from the same industry was added in the current study at ratio of 1:1 by weight. Two different sources of wastewater (Point 1 and Point 2) were used in this study to distinguish the performance of microorganism consortia in both samples. The experiment was conducted by using a lab scale batch anaerobic digester made up from 5L container with separate gas collecting system. The biogas produced was collected by using water displacement method. The experiment was conducted for 30 days and the biogas produced was collected and its volume was recorded at 3 days interval. Based on the data available, wastewater from the first point recorded higher volume of biogas with the total accumulated biogas volume is 216.1 mL. Meanwhile, wastewater sample from Point 2 produced a total of 140.5 mL of biogas, by volume. The data shows that the origin and type of microorganism undeniably play significant role in biogas production. In fact, other factors; pH of wastewater and temperature were also known to affect biogas production. The anaerobic digestion is seen as the promising and sustainable alternatives to current disposal method.

Extraction of bromelain from pineapple peels.

PubMed

Ketnawa, S; Chaiwut, P; Rawdkuen, S

2011-08-01

Large amount of pineapple peels (by-products) is left over after processing and they are a potential source for bromelain extraction. Distilled water (DI), DI containing cysteine and ethylenediaminetetraacetic acid (EDTA) (DI-CE), sodium phosphate buffer pH 7.0 (PB) and PB containing cysteine and EDTA (PB-CE) were used as extractants for bromelain from the pineapple peels. The highest bromelain activity was obtained when it was extracted with PB-CE (867 and 1032 units for Nang Lae and Phu Lae cultv, respectively). The PB could maintain the pH of the extract (pH 5.1-5.7) when compared with others. Under sodium dodecyl sulfate polyacrylamide gel electrophoresis, the extract showed protein bands in the range 24-28 kDa. The protein band with a molecular weight of ∼28 kDa exposed the clear zone on blue background under the casein-substrate gel electrophoresis. The effects of the bromelain extract on the protein patterns of beef, chicken and squid muscles were also determined. Trichloroacetic acid soluble peptide content of all the treated muscles increased when the amount of bromelain extract increased. Decrease in myosin heavy chains and actin was observed in all the muscle types when bromelain extract was used. The best extractant for bromelain from pineapple peels was PB-CE. Moreover, bromelain extract could be used as a muscle food tenderizing agent in food industries.

Effect of vital bleaching with solutions containing different concentrations of hydrogen peroxide and pineapple extract as an additive on human enamel using reflectance spectrophotometer: An in vitro study.

PubMed

Vejai Vekaash, Chitra Janardhanan; Kumar Reddy, Tripuravaram Vinay; Venkatesh, Kondas Vijay

2017-01-01

This study aims to evaluate the color change in human enamel bleached with three different concentrations of hydrogen peroxide, containing pineapple extract as an additive in two different timings, using reflectance spectrophotometer. The study aimed to investigate the bleaching efficacy on natural teeth using natural enzymes. Baseline color values of 10 randomly selected artificially stained incisors were obtained. The specimens were divided into three groups of 20 teeth each: Group 1 - 30% hydrogen peroxide, Group II - 20% hydrogen peroxide, and Group III - 10% hydrogen peroxide. One half of the tooth was bleached with hydrogen peroxide, and other was bleached with hydrogen peroxide and pineapple extract for 20 min (Subgroup A) and 10 min (Subgroup B). The results were statistically analyzed using student's t -test. The mean ΔE values of Group IA (31.62 ± 0.9), Group IIA (29.85 ± 1.2), and Group IIIA (28.65 ± 1.2) showed statistically significant higher values when compared to the mean Δ E values of Group 1A (25.02 ± 1.2), Group IIA (22.86 ± 1.1), and Group IIIA (16.56 ± 1.1). Identical results were obtained in Subgroup B. The addition of pineapple extract to hydrogen peroxide resulted in effective bleaching.

Investigation of Endophytic Bacterial Community in Supposedly Axenic Cultures of Pineapple and Orchids with Evidence on Abundant Intracellular Bacteria.

PubMed

Esposito-Polesi, Natalia Pimentel; de Abreu-Tarazi, Monita Fiori; de Almeida, Cristina Vieira; Tsai, Siu Mui; de Almeida, Marcílio

2017-01-01

Asepsis, defined as the absence of microbial contamination, is one of the most important requirements of plant micropropagation. In long-term micropropagated cultures, there may occasionally occur scattered microorganism growth in the culture medium. These microorganisms are common plant components and are known as latent endophytes. Thus, the aim of this research was to investigate the presence of endophytic bacteria in asymptomatic pineapple and orchid microplants, which were cultivated in three laboratories for 1 year. Isolation and characterization of bacterial isolates, PCR-DGGE from total genomic DNA of microplants and ultrastructural analysis of leaves were performed. In the culture-dependent technique, it was only possible to obtain bacterial isolates from pineapple microplants. In this case, the bacteria genera identified in the isolation technique were Bacillus, Acinetobacter, and Methylobacterium. The scanning electron microscopy and transmission electron microscopy (SEM and TEM) analyses revealed the presence of endophytic bacteria in intracellular spaces in the leaves of pineapple and orchid microplants, independent of the laboratory or cultivation protocol. Our results strongly indicate that there are endophytic bacterial communities inhabiting the microplants before initiation of the in vitro culture and that some of these endophytes persist in their latent form and can also grow in the culture medium even after long-term micropropagation, thus discarding the concept of "truly axenic plants."

Partitioning Carbon Dioxide Emission and Assessing Dissolved Organic Carbon Leaching of a Drained Peatland Cultivated with Pineapple at Saratok, Malaysia

PubMed Central

Lim Kim Choo, Liza Nuriati; Ahmed, Osumanu Haruna

2014-01-01

Pineapples (Ananas comosus (L.) Merr.) cultivation on drained peats could affect the release of carbon dioxide (CO2) into the atmosphere and also the leaching of dissolved organic carbon (DOC). Carbon dioxide emission needs to be partitioned before deciding on whether cultivated peat is net sink or net source of carbon. Partitioning of CO2 emission into root respiration, microbial respiration, and oxidative peat decomposition was achieved using a lysimeter experiment with three treatments: peat soil cultivated with pineapple, bare peat soil, and bare peat soil fumigated with chloroform. Drainage water leached from cultivated peat and bare peat soil was also analyzed for DOC. On a yearly basis, CO2 emissions were higher under bare peat (218.8 t CO2 ha/yr) than under bare peat treated with chloroform (205 t CO2 ha/yr), and they were the lowest (179.6 t CO2 ha/yr) under cultivated peat. Decreasing CO2 emissions under pineapple were attributed to the positive effects of photosynthesis and soil autotrophic activities. An average 235.7 mg/L loss of DOC under bare peat suggests rapid decline of peat organic carbon through heterotrophic respiration and peat decomposition. Soil CO2 emission depended on moderate temperature fluctuations, but it was not affected by soil moisture. PMID:25215335

Pineapple peel wastes as a potential source of antioxidant compounds

NASA Astrophysics Data System (ADS)

Saraswaty, V.; Risdian, C.; Primadona, I.; Andriyani, R.; Andayani, D. G. S.; Mozef, T.

2017-03-01

Indonesia is a large pineapple (Ananas comosus) producing country. Food industries in Indonesia processed this fruit for new products and further resulted wastes of which cause an environmental problems. Approximately, one pineapple fruit total weight is 400 gr of which 60 g is of peel wastes. In order to reduce such pineapple peel wastes (PPW), processing to a valuable product using an environmentally friendly technique is indispensable. PPW contained phenolic compound, ferulic acid, and vitamin A and C as antioxidant. This study aimed to PPW using ethanol and water as well as to analyze its chemical properties. Both dried and fresh PPW were extracted using mixtures of ethanol and water with various concentrations ranging from 15 to 95% (v/v) at room temperature for 24 h. The chemical properties, such as antioxidant activity, total phenolic content (Gallic acid equivalent/GAE), and total sugar content were determined. The results showed that the range of Inhibition Concentration (IC)50 value as antioxidant activity of extracts from dried and fresh PPW were in the range of 0.8±0.05 to 1.3±0.09 mg.mL-1 and 0.25±0.01 to 0.59±0.01 mg.mL-1, respectively, with the highest antioxidant activity was in water extract. The highest of total phenolic content of 0.9 mg.g-1 GAE, was also found in water extract.

Altering histone acetylation status in donor cells with suberoylanilide hydroxamic acid does not affect dog cloning efficiency.

PubMed

Kim, Min Jung; Oh, Hyun Ju; Kim, Geon A; Suh, Han Na; Jo, Young Kwang; Choi, Yoo Bin; Kim, Dong Hoon; Han, Ho Jae; Lee, Byeong Chun

2015-10-15

Although dog cloning technology has been applied to conservation of endangered canids, propagation of elite dogs, and production of transgenic dogs, the efficiency of cloning is still very low. To help overcome this problem, we evaluated the effect of treating donor cells with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on dog cloning efficiency. Relative messenger RNA expressions of the bax1/bcl2 ratio and Dnmt1 in fibroblasts treated with different concentrations (0, 1, 10, 50 μM) of SAHA and durations (0, 20, 44 hours) were compared. Treatment with 1 μM for 20 hours showed significantly lower bax1/bcl2 and Dnmt1 transcript abundance. Acetylation of H3K9 was significantly increased after SAHA treatment, but H4K5, H4K8 and H4K16 were not changed. After SCNT using control or donor cells treated with SAHA, a total of 76 and 64 cloned embryos were transferred to seven and five recipients, respectively. Three fetuses were diagnosed in both control and SAHA-treated groups by ultrasonography 29 days after the embryo transfer, but there was no significant difference in the pregnancy rate (4.2% vs. 4.3%). In conclusion, although SAHA treatment as used in this study significantly decreased bax1/bcl2 and Dnmt1 transcripts of donor nuclei, as well as increased H3 acetylation, it was not enough to increase in vivo developmental competence of cloned dog embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

Generation and functional characterization of a clonal murine periportal Kupffer cell line from H-2Kb -tsA58 mice.

PubMed

Dory, Daniel; Echchannaoui, Hakim; Letiembre, Maryse; Ferracin, Fabrizia; Pieters, Jean; Adachi, Yoshiyuki; Akashi, Sachiko; Zimmerli, Werner; Landmann, Regine

2003-07-01

Murine Kupffer cells (KCs) are heterogeneous and survive only for a short time in vitro. Here, a clonal, murine KC line was generated from transgenic mice, expressing the thermolabile mutant tsA58 of the Simian virus 40 large T antigen under the control of the H-2K(b) promoter. Thirty-three degrees Celsius and 37 degrees C but not 39 degrees C have been permissive for growth of the clone; it required conditioned media from hepatocytes and endothelial cells for proliferation. In contrast to primary cells, the cells of the clone were uniform, survived detachment, and could therefore be analyzed by cytofluorimetry. The clone, as primary KCs, constitutively expressed nonspecific esterase, peroxidase, MOMA-2, BM8, scavenger receptor A, CD14, and Toll-like receptor 4 (TLR4); the antigen-presenting molecules CD40, CD80, and CD1d; and endocytosed dextran-fluorescein isothiocyanate. It lacked complement, Fc receptors, F4/80 marker, and the phagosomal coat protein tryptophan aspartate-containing coat protein (TACO). The clone exhibited CD14- and TLR4/MD2-independent, plasma-dependent lipopolysaccharide (LPS) binding, Escherichia coli and Streptococcus pneumoniae phagocytosis, and LPS- and interferon-gamma-induced NO production but no tumor necrosis factor alpha, interleukin (IL)-6, or IL-10 release. The large size, surface-marker expression, and capacity to clear gram-negative and -positive bacteria indicate that the clone was derived from the periportal, large KC subpopulation. The clone allows molecular studies of anti-infective and immune functions of KCs.

Utilization of wastewater originated from naturally fermented virgin coconut oil manufacturing process for bioextract production: physico-chemical and microbial evolution.

PubMed

Tripetchkul, Sudarut; Kusuwanwichid, Sasithorn; Koonsrisuk, Songpon; Akeprathumchai, Saengchai

2010-08-01

Production of virgin coconut oil via natural fermentation has led to large amount of wastes being generated, i.e., coconut pulp and wastewater containing coconut crème. Objective of this study is to gain more insight into the feasibility of utilization of such wastes as raw materials together with several types of wastes such as fish waste and/or pineapple peel for bioextract production. Chemical, physico-chemical and biological changes including phytotoxicity of the fermented mixture were closely monitored. Physical observation suggested that fermentation of bioextract obtained with fish waste appeared to be complete within the first month of fermentation while bioextract obtained using pineapple waste seemed to be complete after 8 months post-fermentation. Fermentation broth is of blackish color with alcoholic as well as acidic odour with no gas bubble and/or yeast film present on top of the surface. During the whole fermentation interval, several attributes of both bioextracts, e.g., pH, chemical oxygen demand (COD) and organic acids, were statistically different. Further, the total bacteria and lactic acid bacteria present in pineapple bioextract were statistically higher than those of the fish bioextract (p<0.01). The highest germination indices of 123 and 106 were obtained at 21 and 14 days post-fermentation for fish and pineapple bioextracts, respectively. In addition, qualities of both bioextracts conformed well with those specified by the Thai standard for liquid biofertilizer after 1 month fermentation. Results further showed that wastewater derived from virgin coconut oil manufacturing process could effectively be employed together with other types of wastes such as fish waste and pineapple peel for bioextract production. However, for the best bioextract quality, fermentation should be carefully planned since over fermentation led to bioextract of low qualities. Copyright 2010 Elsevier Ltd. All rights reserved.

Uncertainty in recharge estimation: impact on groundwater vulnerability assessments for the Pearl Harbor Basin, O'ahu, Hawai'i, U.S.A.

NASA Astrophysics Data System (ADS)

Giambelluca, Thomas W.; Loague, Keith; Green, Richard E.; Nullet, Michael A.

1996-06-01

In this paper, uncertainty in recharge estimates is investigated relative to its impact on assessments of groundwater contamination vulnerability using a relatively simple pesticide mobility index, attenuation factor (AF). We employ a combination of first-order uncertainty analysis (FOUA) and sensitivity analysis to investigate recharge uncertainties for agricultural land on the island of O'ahu, Hawai'i, that is currently, or has been in the past, under sugarcane or pineapple cultivation. Uncertainty in recharge due to recharge component uncertainties is 49% of the mean for sugarcane and 58% of the mean for pineapple. The components contributing the largest amounts of uncertainty to the recharge estimate are irrigation in the case of sugarcane and precipitation in the case of pineapple. For a suite of pesticides formerly or currently used in the region, the contribution to AF uncertainty of recharge uncertainty was compared with the contributions of other AF components: retardation factor (RF), a measure of the effects of sorption; soil-water content at field capacity (ΘFC); and pesticide half-life (t1/2). Depending upon the pesticide, the contribution of recharge to uncertainty ranks second or third among the four AF components tested. The natural temporal variability of recharge is another source of uncertainty in AF, because the index is calculated using the time-averaged recharge rate. Relative to the mean, recharge variability is 10%, 44%, and 176% for the annual, monthly, and daily time scales, respectively, under sugarcane, and 31%, 112%, and 344%, respectively, under pineapple. In general, uncertainty in AF associated with temporal variability in recharge at all time scales exceeds AF. For chemicals such as atrazine or diuron under sugarcane, and atrazine or bromacil under pineapple, the range of AF uncertainty due to temporal variability in recharge encompasses significantly higher levels of leaching potential at some locations than that indicated by the AF estimate.

Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease.

PubMed

Liu, Xin; Yang, Lihua; Zhou, Xianyao; Zhou, Miaoping; Lu, Yan; Ma, Lingjian; Ma, Hongxiang; Zhang, Zengyan

2013-05-01

The disease take-all, caused by the fungus Gaeumannomyces graminis, is one of the most destructive root diseases of wheat worldwide. Breeding resistant cultivars is an effective way to protect wheat from take-all. However, little progress has been made in improving the disease resistance level in commercial wheat cultivars. MYB transcription factors play important roles in plant responses to environmental stresses. In this study, an R2R3-MYB gene in Thinopyrum intermedium, TiMYB2R-1, was cloned and characterized. The gene sequence includes two exons and an intron. The expression of TiMYB2R-1 was significantly induced following G. graminis infection. An in vitro DNA binding assay proved that TiMYB2R-1 protein could bind to the MYB-binding site cis-element ACI. Subcellular localization assays revealed that TiMYB2R-1 was localized in the nucleus. TiMYB2R-1 transgenic wheat plants were generated, characterized molecularly, and evaluated for take-all resistance. PCR and Southern blot analyses confirmed that TiMYB2R-1 was integrated into the genomes of three independent transgenic wheat lines by distinct patterns and the transgene was heritable. Reverse transcription-PCR and western blot analyses revealed that TiMYB2R-1 was highly expressed in the transgenic wheat lines. Based on disease response assessments for three successive generations, the significantly enhanced resistance to take-all was observed in the three TiMYB2R-1-overexpressing transgenic wheat lines. Furthermore, the transcript levels of at least six wheat defence-related genes were significantly elevated in the TiMYB2R-1 transgenic wheat lines. These results suggest that engineering and overexpression of TiMYB2R-1 may be used for improving take-all resistance of wheat and other cereal crops.

Salt tolerance and activity of antioxidative enzymes of transgenic finger millet overexpressing a vacuolar H(+)-pyrophosphatase gene (SbVPPase) from Sorghum bicolor.

PubMed

Anjaneyulu, Ediga; Reddy, Palle Surender; Sunita, Merla Srilakshmi; Kishor, Polavarapu B Kavi; Meriga, Balaji

2014-06-15

A vacuolar proton pyrophosphatase cDNA clone was isolated from Sorghum bicolor (SbVPPase) using end-to-end gene-specific primer amplification. It showed 80-90% homology at the nucleotide and 85-95% homology at the amino acid level with other VPPases. The gene was introduced into expression vector pCAMBIA1301 under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter and transformed into Agrobacterium tumifaciens strain LBA4404 to infect embryogenic calli of finger millet (Eleusine coracana). Successful transfer of SbVPPase was confirmed by a GUS histochemical assay and PCR analysis. Both, controls and transgenic plants were subjected to 100 and 200mM NaCl and certain biochemical and physiological parameters were studied. Relative water content (RWC), plant height, leaf expansion, finger length and width and grain weight were severely reduced (50-70%), and the flowering period was delayed by 20% in control plants compared to transgenic plants under salinity stress. With increasing salt stress, the proline and chlorophyll contents as well as the enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) increased by 25-100% in transgenics, while malondialdehyde (MDA) showed a 2-4-fold decrease. The increased activities of antioxidant enzymes and the reduction in the MDA content suggest efficient scavenging of reactive oxygen species (ROS) in transgenics and, as a consequence, probably alleviation of salt stress. Also, the leaf tissues of the transgenics accumulated 1.5-2.5-fold higher Na(+) and 0.4-0.8-fold higher K(+) levels. Together, these results clearly demonstrate that overexpression of SbVPPase in transgenic finger millet enhances the plant's performance under salt stress. Copyright © 2014 Elsevier GmbH. All rights reserved.

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector.

PubMed

Do, Ltk; Wittayarat, M; Terazono, T; Sato, Y; Taniguchi, M; Tanihara, F; Takemoto, T; Kazuki, Y; Kazuki, K; Oshimura, M; Otoi, T

2016-12-01

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 μs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 μs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells. © 2016 Blackwell Verlag GmbH.

Self-Cloning CRISPR.

PubMed

Arbab, Mandana; Sherwood, Richard I

2016-08-17

CRISPR/Cas9-gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self-cloning CRISPR/Cas9 (scCRISPR) uses a self-cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR-amplified site-specific sgRNA sequences within the target cell by homologous recombination to circumvent the process of sgRNA plasmid construction. Through this mechanism, scCRISPR enables gene editing within 2 hr once sgRNA oligos are available, with high efficiency equivalent to conventional sgRNA targeting: >90% gene knockout in both mouse and human embryonic stem cells and cancer cell lines. Furthermore, using PCR-based addition of short homology arms, we achieve efficient site-specific knock-in of transgenes such as GFP without traditional plasmid cloning or genome-integrated selection cassette (2% to 4% knock-in rate). The methods in this paper describe the most rapid and efficient means of CRISPR gene editing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

The Coast Artillery Journal. Volume 74, Number 2, February 1931

DTIC Science & Technology

1931-02-01

Varieties of Palms Near Honolulu l,ci womell at the steamcl’S alld workmell with flower ’Heaths around thcir hats nl’C casil~’ picked out. ~ot so easil...add yariety and thrills as well. The sugar cane of the lowlands giyes \\ray to pineapple plantations in the red dirt for which Schofield is famou,s...Hiding Club. Hides through the deep gulches, the pineapple fields, and the guaya thickets of the uplands are welI attended. In the Spring the Diyision

Comparative study of two methods of fractionation bromelain from pineapple core extract (Ananas comosus)

NASA Astrophysics Data System (ADS)

Febriani, K.; Wahyuni, I.; Setiasih, S.; Hudiyono, S.

2017-07-01

The enzyme can be purified by fractional precipitation. This can be done by salt or organic solvent. In this research, purification of bromelain from pineapple core by fractional precipitation was done by 2 compounds, ammonium sulfate, and ethanol. Fractional precipitation by ammonium sulfate proved to be more effective as it yielded a higher specific activity. Specific activity by ethanol and ammonium sulfate is 4.6480 U/mg at 0-60 % saturation and 8.2243 U/mg at 50-80 % saturation.

Monitoring the efficacy of mutated Allium sativum leaf lectin in transgenic rice against Rhizoctonia solani.

PubMed

Ghosh, Prithwi; Sen, Senjuti; Chakraborty, Joydeep; Das, Sampa

2016-03-01

Rice sheath blight, caused by Rhizoctonia solani is one of the most devastating diseases of rice. It is associated with significant reduction in rice productivity worldwide. A mutant variant of mannose binding Allium sativum leaf agglutinin (mASAL) was previously reported to exhibit strong antifungal activity against R. solani. In this study, the mASAL gene has been evaluated for its in planta antifungal activity in rice plants. mASAL was cloned into pCAMBIA1301 binary vector under the control of CaMV35S promoter. It was expressed in an elite indica rice cv. IR64 by employing Agrobacterium tumefaciens-mediated transformation. Molecular analyses of transgenic plants confirmed the presence and stable integration of mASAL gene. Immunohistofluorescence analysis of various tissue sections of plant parts clearly indicated the constitutive expression of mASAL. The segregation pattern of mASAL transgene was observed in T1 progenies in a 3:1 Mendelian ratio. The expression of mASAL was confirmed in T0 and T1 plants through western blot analysis followed by ELISA. In planta bioassay of transgenic lines against R. solani exhibited an average of 55 % reduction in sheath blight percentage disease index (PDI). The present study opens up the possibility of engineering rice plants with the antifungal gene mASAL, conferring resistance to sheath blight.

ABI-like transcription factor gene TaABL1 from wheat improves multiple abiotic stress tolerances in transgenic plants.

PubMed

Xu, Dong-Bei; Gao, Shi-Qing; Ma, You-Zhi; Xu, Zhao-Shi; Zhao, Chang-Ping; Tang, Yi-Miao; Li, Xue-Yin; Li, Lian-Cheng; Chen, Yao-Feng; Chen, Ming

2014-12-01

The phytohormone abscisic acid (ABA) plays crucial roles in adaptive responses of plants to abiotic stresses. ABA-responsive element binding proteins (AREBs) are basic leucine zipper transcription factors that regulate the expression of downstream genes containing ABA-responsive elements (ABREs) in promoter regions. A novel ABI-like (ABA-insensitive) transcription factor gene, named TaABL1, containing a conserved basic leucine zipper (bZIP) domain was cloned from wheat. Southern blotting showed that three copies were present in the wheat genome. Phylogenetic analyses indicated that TaABL1 belonged to the AREB subfamily of the bZIP transcription factor family and was most closely related to ZmABI5 in maize and OsAREB2 in rice. Expression of TaABL1 was highly induced in wheat roots, stems, and leaves by ABA, drought, high salt, and low temperature stresses. TaABL1 was localized inside the nuclei of transformed wheat mesophyll protoplast. Overexpression of TaABL1 enhanced responses of transgenic plants to ABA and hastened stomatal closure under stress, thereby improving tolerance to multiple abiotic stresses. Furthermore, overexpression of TaABL1 upregulated or downregulated the expression of some stress-related genes controlling stomatal closure in transgenic plants under ABA and drought stress conditions, suggesting that TaABL1 might be a valuable genetic resource for transgenic molecular breeding.

Cellular Localization of Wheat High Molecular Weight Glutenin Subunits in Transgenic Rice Grain

PubMed Central

Jo, Yeong-Min; Cho, Kyoungwon; Lee, Hye-Jung; Lim, Sun-Hyung; Kim, Jin Sun; Kim, Young-Mi; Lee, Jong-Yeol

2017-01-01

Rice (Oryza sativa L.) is a primary global food cereal. However, when compared to wheat, rice has poor food processing qualities. Dough that is made from rice flour has low viscoelasticity because rice seed lacks storage proteins that are comparable to gluten protein from wheat. Thus, current research efforts aim to improve rice flour processing qualities through the transgenic expression of viscoelastic proteins in rice seeds. In this study, we characterized the transgenic expression of wheat glutenin subunits in rice seeds. The two genes 1Dx5_KK and 1Dy10_JK, which both encode wheat high-molecular-weight glutenin subunits that confer high dough elasticity, were cloned from Korean wheat cultivars KeumKang and JoKyung, respectively. These genes were inserted into binary vectors under the control of the rice endosperm-specific Glu-B1 promoter and were expressed in the high-amylose Korean rice cultivar Koami (Oryza sativa L.). Individual expression of both glutenin subunits was confirmed by SDS-PAGE and immunoblot analyses performed using T3 generation of transgenic rice seeds. The subcellular localization of 1Dx5_KK and 1Dy10_JK in the rice seed endosperm was confirmed by immunofluorescence analysis, indicating that the wheat glutenin subunits accumulate in protein body-II and novel protein body types in the rice seed. These results contribute to our understanding of engineered seed storage proteins in rice. PMID:29156580

AgFNS overexpression increase apigenin and decrease anthocyanins in petioles of transgenic celery.

PubMed

Tan, Guo-Fei; Ma, Jing; Zhang, Xin-Yue; Xu, Zhi-Sheng; Xiong, Ai-Sheng

2017-10-01

Apigenin and anthocyanin biosyntheses share common precursors in plants. Flavone synthase (FNS) converts naringenin into apigenin in higher plants. Celery is an important edible and medical vegetable crop that contains apigenin in its tissues. However, the effect of high AgFNS gene expression on the apigenin and anthocyanins contents of purple celery remains to be elucidated. In this study, the AgFNS gene was cloned from purple celery ('Nanxuan liuhe purple celery') and overexpressed in this purple celery to determine its influence on anthocyanins and apigenin contents. Results showed that the AgFNS gene was 1068bp, which encodes 355 amino acid residues. Evolution analysis showed that the AgFNS protein belongs to the FSN I type. In AgFNS transgenic celery, the anthocyanins content in petioles was lower than that wild-type celery plants. Apigenin content increased in the petioles of AgFNS transgenic celery. The transcript levels of the AgPAL, AgC4H, AgCHS, and AgCHI genes were up-regulated, whereas those of the AgF3H, AgF3'H, AgDFR, AgANS, and Ag3GT genes were down-regulated in the petioles of AgFNS transgenic plants compared with wild-type celery plants. This work provides basic knowledge about the function of the AgFNS gene in the anthocyanin and apigenin biosyntheses of celery. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhanced tolerance and remediation to mixed contaminates of PCBs and 2,4-DCP by transgenic alfalfa plants expressing the 2,3-dihydroxybiphenyl-1,2-dioxygenase.

PubMed

Wang, Yan; Ren, Hejun; Pan, Hongyu; Liu, Jinliang; Zhang, Lanying

2015-04-09

Polychlorinated biphenyls (PCBs) and 2,4-dichlorophenol (2,4-DCP) generally led to mixed contamination of soils as a result of commercial and agricultural activities. Their accumulation in the environment poses great risks to human and animal health. Therefore, the effective strategies for disposal of these pollutants are urgently needed. In this study, genetic engineering to enhance PCBs/2,4-DCP phytoremediation is a focus. We cloned the 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC.B) from a soil metagenomic library, which is the key enzyme of aerobic catabolism of a variety of aromatic compounds, and then it was expressed in alfalfa driven by CaMV 35S promoter using Agrobacterium-mediated transformation. Transgenic line BB11 was selected out through PCR, Western blot analysis and enzyme activity assays. Its disposal and tolerance to both PCBs and 2,4-DCP were examined. The tolerance capability of transgenic line BB11 towards complex contaminants of PCBs/2,4-DCP significantly increased compared with non-transgenic plants. Strong dissipation of PCBs and high removal efficiency of 2,4-DCP were exhibited in a short time. It was confirmed expressing BphC.B would be a feasible strategy to help achieving phytoremediation in mixed contaminated soils with PCBs and 2,4-DCP. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of the CAD gene from Eucalyptus urophylla GLU4 and its functional analysis in transgenic tobacco.

PubMed

Chen, B W; Xiao, Y F; Li, J J; Liu, H L; Qin, Z H; Gai, Y; Jiang, X N

2016-12-02

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in lignin biosynthesis. The genus Eucalyptus belongs to the family Myrtaceae, which is the main cultivated species in China. Eucalyptus urophylla GLU4 (GLU4) is widely grown in Guangxi. It is preferred for pulping because of its excellent cellulose content and fiber length. Based on GLU4 and CAD gene expression, a Eucalyptus variety low in lignin content should be obtained using transgenic technology, which could reduce the cost of pulp and improve the pulping rate, and have favorable prospects for application. However, the role and function of CAD in GLU4 is still unclear. In the present study, EuCAD was cloned from GLU4 and identified using bioinformatic tools. Subsequently, in order to evaluate its impact on lignin synthesis, a full-length EuCAD RNAi vector was constructed, and transgenic tobacco was obtained via Agrobacterium-mediated transformation. A significant decrease in CAD expression and lignin content in transgenic tobacco demonstrated a key role for EuCAD in lignin biosynthesis and established a regulatory role for RNAi. In our study, the direct molecular basis of EuCAD expression was determined, and the potential regulatory effects of this RNAi vector on lignin biosynthesis in E. urophylla GLU4 were demonstrated. Our results provide a theoretical basis for the study of lignin biosynthesis in Eucalyptus.

Antisense expression of the fasciclin-like arabinogalactan protein FLA6 gene in Populus inhibits expression of its homologous genes and alters stem biomechanics and cell wall composition in transgenic trees.

PubMed

Wang, Haihai; Jiang, Chunmei; Wang, Cuiting; Yang, Yang; Yang, Lei; Gao, Xiaoyan; Zhang, Hongxia

2015-03-01

Fasciclin-like arabinogalactan proteins (FLAs) play important roles in the growth and development of roots, stems, and seeds in Arabidopsis. However, their biological functions in woody plants are largely unknown. In this work, we investigated the possible function of PtFLA6 in poplar. Quantitative real-time PCR, PtFLA6-yellow fluorescent protein (YFP) fusion protein subcellular localization, Western blotting, and immunohistochemical analyses demonstrated that the PtFLA6 gene was expressed specifically in the xylem of mature stem, and PtFLA6 protein was distributed ubiquitous in plant cells and accumulated predominantly in stem xylem fibres. Antisense expression of PtFLA6 in the aspen hybrid clone Poplar davidiana×Poplar bolleana reduced the transcripts of PtFLA6 and its homologous genes. Transgenic plants that showed a significant reduction in the transcripts of PtFLAs accumulated fewer PtFLA6 and arabinogalactan proteins than did the non-transgenic plants, leading to reduced stem flexural strength and stiffness. Further studies revealed that the altered stem biomechanics of transgenic plants could be attributed to the decreased cellulose and lignin composition in the xylem. In addition expression of some xylem-specific genes involved in cell wall biosynthesis was downregulated in these transgenic plants. All these results suggest that engineering the expression of PtFLA6 and its homologues could modulate stem mechanical properties by affecting cell wall composition in trees. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

RAPD and SSR Polymorphisms in Mutant Lines of Transgenic Wheat Mediated by Low Energy Ion Beam

NASA Astrophysics Data System (ADS)

Wang, Tiegu; Huang, Qunce; Feng, Weisen

2007-10-01

Two types of markers-random amplified polymorphic DNA (RAPD) and simple sequence repeat DNA (SSR)-have been used to characterize the genetic diversity among nine mutant lines of transgenic wheat intermediated by low energy ion beam and their four receptor cultivars. The objectives of this study were to analyze RAPD-based and SSR-based genetic variance among transgenic wheat lines and with their receptors, and to find specific genetic markers of special traits of transgenic wheat lines. 170 RAPD primers were amplified to 733 fragments in all the experimental materials. There were 121 polymorphic fragments out of the 733 fragments with a ratio of polymorphic fragments of 16.5%. 29 SSR primer pairs were amplified to 83 fragments in all the experiment materials. There were 57 polymorphic fragments out of the 83 fragments with a ratio of polymorphic fragments of 68.7%. The dendrograms were prepared based on a genetic distance matrix using the UPGMA (Unweighted Pair-group Method with Arithmetic averaging) algorithm, which corresponded well to the results of the wheat pedigree analysis and separated the 13 genotypes into four groups. Association analysis between RAPD and SSR markers with the special traits of transgenic wheat mutant lines discovered that three RAPD markers, s1, opt-16, and f14, were significantly associated with the muticate trait, while three SSR markers, Rht8 (Xgwm261), Rht-B1b, and Rht-D1b, highly associated with the dwarf trait. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.

Alu sequence involvement in transcriptional insulation of the keratin 18 gene in transgenic mice.

PubMed Central

Thorey, I S; Ceceña, G; Reynolds, W; Oshima, R G

1993-01-01

The human keratin 18 (K18) gene is expressed in a variety of adult simple epithelial tissues, including liver, intestine, lung, and kidney, but is not normally found in skin, muscle, heart, spleen, or most of the brain. Transgenic animals derived from the cloned K18 gene express the transgene in appropriate tissues at levels directly proportional to the copy number and independently of the sites of integration. We have investigated in transgenic mice the dependence of K18 gene expression on the distal 5' and 3' flanking sequences and upon the RNA polymerase III promoter of an Alu repetitive DNA transcription unit immediately upstream of the K18 promoter. Integration site-independent expression of tandemly duplicated K18 transgenes requires the presence of either an 825-bp fragment of the 5' flanking sequence or the 3.5-kb 3' flanking sequence. Mutation of the RNA polymerase III promoter of the Alu element within the 825-bp fragment abolishes copy number-dependent expression in kidney but does not abolish integration site-independent expression when assayed in the absence of the 3' flanking sequence of the K18 gene. The characteristics of integration site-independent expression and copy number-dependent expression are separable. In addition, the formation of the chromatin state of the K18 gene, which likely restricts the tissue-specific expression of this gene, is not dependent upon the distal flanking sequences of the 10-kb K18 gene but rather may depend on internal regulatory regions of the gene. Images PMID:7692231

Helper-Dependent Adenoviral Vectors.

PubMed

Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

2011-10-29

Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology.

Helper-Dependent Adenoviral Vectors

PubMed Central

Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

2012-01-01

Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology. PMID:24533227

Bromelain enzyme from pineapple: in vitro activity study under different micropropagation conditions.

PubMed

Vilanova Neta, Jaci Lima; da Silva Lédo, Ana; Lima, Aloisio André Bonfim; Santana, José Carlos Curvelo; Leite, Nadjma Souza; Ruzene, Denise Santos; Silva, Daniel Pereira; de Souza, Roberto Rodrigues

2012-09-01

The aim of this work was to evaluate the activity of bromelain in pineapple plants (Ananas comosus var. Comosus), Pérola cultivar, produced in vitro in different culture conditions. This enzyme, besides its pharmacological effects, is also employed in food industries, such as breweries and meat processing. In this work, the enzymatic activity was evaluated in the tissues of leaves and stems of plants grown in culture medium without plant growth regulator. The most significant levels of bromelain were observed in leaf tissue after 4 months of culture in vitro in medium with a filter paper bridge, followed by medium gelled by the agar. The results of this study, regarding the different structures of the pineapple (leaves and stems) in vitro showed that the activity of bromelain varied depending on the culture conditions, the time and structure of which was quantified, ensuring a viable strategy in the production of seedlings with high levels of bromelain in subsequent phases of micropropagation.

Postprandial glucose and insulin responses to various tropical fruits of equivalent carbohydrate content in non-insulin-dependent diabetes mellitus.

PubMed

Roongpisuthipong, C; Banphotkasem, S; Komindr, S; Tanphaichitr, V

1991-11-01

The plasma glucose and insulin responses were determined in 10 NIDDM female patients following the ingestion of tropical fruit containing 25 g of carbohydrate. The five tropical fruits were pineapple, mango, banana, durian and rambutan. Blood was drawn at 0, 30, 60, 120 and 180 min, respectively. The results showed that the glucose-response curves to mango and banana were significantly less than those to rambutan, durian and pineapple (P less than 0.05). Only the glucose area after mango ingestion was significantly less than the glucose areas of the other fruits (P less than 0.05). The insulin response curve and insulin area after durian ingestion was statistically greater than after ingestion of the others. We concluded that after mango ingestion, the glucose area was lower than it had been after rambutan, durian and pineapple ingestion and the insulin area was lower than that after durian ingestion of equivalent carbohydrate content in type 2 (NIDDM) diabetes.

An Electrochemical Impedance Spectroscopy-Based Technique to Identify and Quantify Fermentable Sugars in Pineapple Waste Valorization for Bioethanol Production

PubMed Central

Conesa, Claudia; García-Breijo, Eduardo; Loeff, Edwin; Seguí, Lucía; Fito, Pedro; Laguarda-Miró, Nicolás

2015-01-01

Electrochemical Impedance Spectroscopy (EIS) has been used to develop a methodology able to identify and quantify fermentable sugars present in the enzymatic hydrolysis phase of second-generation bioethanol production from pineapple waste. Thus, a low-cost non-destructive system consisting of a stainless double needle electrode associated to an electronic equipment that allows the implementation of EIS was developed. In order to validate the system, different concentrations of glucose, fructose and sucrose were added to the pineapple waste and analyzed both individually and in combination. Next, statistical data treatment enabled the design of specific Artificial Neural Networks-based mathematical models for each one of the studied sugars and their respective combinations. The obtained prediction models are robust and reliable and they are considered statistically valid (CCR% > 93.443%). These results allow us to introduce this EIS-based technique as an easy, fast, non-destructive, and in-situ alternative to the traditional laboratory methods for enzymatic hydrolysis monitoring. PMID:26378537

Inactivation of the microbiota and effect on the quality attributes of pineapple juice using a continuous flow ultrasound-assisted supercritical carbon dioxide system.

PubMed

Paniagua-Martínez, I; Mulet, A; García-Alvarado, M A; Benedito, J

2018-01-01

Supercritical carbon dioxide inactivation technology represents a promising nonthermal processing method, as it causes minimum impact on the nutritional food properties. The aim of this study was to analyze the combined effect of supercritical carbon dioxide and high-power ultrasound on the inactivation of natural microbiota and the quality attributes of pineapple juice treated in a continuous flow system. Different juice residence times (3.06-4.6 min), at 100 bar and 31.5 ℃, were used. The results indicated that the microbiota inactivation was complete and the differences obtained in the quality attributes (2.2% for pH, 4.8% for °Brix, 2% for vitamin C) were minimal. During storage, microorganisms were not able to recover and the vitamin C decrease could be limited to 8.2% after four weeks. The results demonstrated that the supercritical carbon dioxide-high-power ultrasound technique could be an excellent alternative for the cold pasteurization of pineapple juice.

Influence of the fiber from agro-industrial co-products as functional food ingredient on the acceptance, neophobia and sensory characteristics of cooked sausages.

PubMed

Díaz-Vela, Juan; Totosaus, Alfonso; Escalona-Buendía, Héctor B; Pérez-Chabela, M Lourdes

2017-02-01

The sensory analysis of new products is essential for subsequent acceptance by consumers, moreover in the functional food market. The acceptance and food neophobia of cooked sausages formulated with cactus pear fiber or pineapple pear fiber, as functional ingredient, was complemented with a sensory characterization by R-index and qualitative descriptive analysis (QDA). Female consumers aged between 40 and 50 years showed greater interest in the consumption of healthy foods, with a higher level of food neophobia towards pineapple fiber sausages. R-index for taste was higher in pineapple fiber samples. Cactus pear fiber samples presented higher R-index score for texture. In QDA, color, sweet, astringent and bitter flavors, pork meat smell and a firm and plastic texture were significant, with a good relationship (38%) between the evaluated attributes. Sensory attributes are important on the acceptance and neophobia of functional foods like cooked sausages with fruit peel fiber as functional ingredient.

Polyphenol oxidase and peroxidase expression in four pineapple varieties (Ananas comosus L.) after a chilling injury.

PubMed

Raimbault, Astrid-Kim; Marie-Alphonsine, Paul-Alex; Horry, Jean-Pierre; Francois-Haugrin, Madlyn; Romuald, Karell; Soler, Alain

2011-01-12

Pineapple internal browning (IB) is a chilling injury that produces enzymatic browning associated with flesh translucency. Pineapple biodiversity allowed the investigation of how polyphenol oxidase (PPO) and peroxidase (POD) activities with their different isoforms are involved in the IB mechanism. Fruits of four varieties that expressed IB symptoms differently, Smooth Cayenne (SCay) and the hybrids MD2, Flhoran 41 (Flh 41), and Flhoran 53 (Flh 53), were stressed by cold. The susceptible varieties showed classical brown spots but different patterns of IB, whereas MD2 and controls showed no IB. Enzymatic activities were measured on fruit protein extracts and PPO and POD isoforms separated on mini-gels (PhastSystem). Only PPO activity was significantly enhanced in the presence of IB. Up to six PPO isoforms were identified in the susceptible varieties. PPO was barely detectable in the nonsusceptible variety MD2 and in controls. The number of PPO isoforms and the total PPO activity after chilling are varietal characteristics.

Sulfur-containing constituents and one 1H-pyrrole-2-carboxylic acid derivative from pineapple [Ananas comosus (L.) Merr.] fruit.

PubMed

Zheng, Zong-Ping; Ma, Jinyu; Cheng, Ka-Wing; Chao, Jianfei; Zhu, Qin; Chang, Raymond Chuen-Chung; Zhao, Ming; Lin, Zhi-Xiu; Wang, Mingfu

2010-12-01

Two sulfur-containing compounds, (S)-2-amino-5-((R)-1-carboxy-2-((E)-3-(4-hydroxy-3-methoxyphenyl)allylthio)ethyl-amino)-5-oxopentanoic acid (1) and (S)-2-amino-5-((R)-1-(carboxymethylamino)-3-((E)-3-(4-hydroxyphenyl)allylthio)-1-oxopropan-2-ylamino)-5-oxopentanoic acid (2), and one 1H-pyrrole-2-carboxylic acid derivative, 6-(3-(1H-pyrrole-2-carbonyloxy)-2-hydroxypropoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-carboxylic acid (3), together with eighteen known phenolic compounds, were isolated from the fruits of pineapple. Their structures were elucidated by a combination of spectroscopic analyses. Some of these compounds showed inhibitory activities against tyrosinase. The half maximal inhibitory concentration values of compounds 1, 4, 5, 6, 7 are lower than 1 mM. These compounds may contribute to the well-known anti-browning effect of pineapple juice and be potential skin whitening agents in cosmetic applications. Copyright © 2010 Elsevier Ltd. All rights reserved.

The biochemical characterization, stabilization studies and the antiproliferative effect of bromelain against B16F10 murine melanoma cells.

PubMed

São Paulo Barretto Miranda, Íngara Keisle; Fontes Suzart Miranda, Anderson; Souza, Fernanda Vidigal Duarte; Vannier-Santos, Marcos André; Pirovani, Carlos Priminho; Pepe, Iuri Muniz; Rodowanski, Ivanoé João; Ferreira, Katiúcia Tícila de Souza Eduvirgens; Mendes Souza Vaz, Luciano; de Assis, Sandra Aparecida

2017-06-01

The current study aims to extract bromelain from different parts (stem, crown, peels, pulp and leaves) of Ananas comosus var. comosus AGB 772; to determine of optimum pH and temperature; to test bromelain stability in disodium EDTA and sodium benzoate, and to investigate its pharmacological activity on B16F10 murine melanoma cells in vitro. The highest enzymatic activity was found in bromelain extracted from the pulp and peel. The optimum bromelain pH among all studied pineapple parts was 6.0. The optimum temperature was above 50 °C in all bromelain extracts. The fluorescence analysis confirmed the stability of bromelain in the presence of EDTA and sodium benzoate. Bromelain was pharmacologically active against B16F10 melanoma cells and it was possible verifying approximately 100% inhibition of tumor cell proliferation in vitro. Since bromelain activity was found in different parts of pineapple plants, pineapple residues from the food industry may be used for bromelain extraction.

Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties.

PubMed

Qiao, Junhua; Oumard, André; Wegloehner, Wolfgang; Bode, Juergen

2009-07-24

Site-specific recombinases have revolutionized the systematic generation of transgenic cell lines and embryonic stem cells/animals and will ultimately also reveal their potential in the genetic modification of induced pluripotent stem cells. Introduced in 1994, our Flp recombinase-mediated cassette exchange strategy permits the exchange of a target cassette for a cassette with the gene of interest, introduced as a part of an exchange vector. The process is "clean" in the sense that it does not co-introduce prokaryotic vector parts; neither does it leave behind a selection marker. Stringent selection principles provide master cell lines permitting subsequent recombinase-mediated cassette exchange cycles in the absence of a drug selection and with a considerable efficiency (approximately 10%). Exemplified by Chinese hamster ovary cells, the strategy proves to be successful even for cell lines with an unstable genotype.

Construction of a standard reference plasmid containing seven target genes for the detection of transgenic cotton.

PubMed

Wang, Xujing; Tang, Qiaoling; Dong, Lei; Dong, Yufeng; Su, Yueyan; Jia, Shirong; Wang, Zhixing

2014-07-01

Insect resistance and herbicide tolerance are the dominant traits of commercialized transgenic cotton. In this study, we constructed a general standard reference plasmid for transgenic cotton detection. Target genes, including the cowpea trypsin gene cptI, the insect resistance gene cry1Ab/1Ac, the herbicide tolerance gene cp4-epsps, the Agrobacterium tumefaciens nopaline synthase (Nos) terminator that exists in transgenic cotton and part of the endogenous cotton SadI gene were amplified from plasmids pCPT1, pBT, pCP4 and pBI121 and from DNA of the nontransgenic cotton line K312, respectively. The genes cry1Ab/1Ac and cptI, as well as cp4-epsps and the Nos terminator gene, were ligated together to form the fusion genes cptI-Bt and cp4-Nos, respectively, by overlapping PCR. We checked the validity of genes Sad1, cptI-Bt and cp4-Nos by DNA sequencing. Then, positive clones of cptI-Bt, cp4-Nos and Sad1 were digested with the corresponding restriction enzymes and ligated sequentially into vector pCamBIA2300, which contains the CAMV 35S promoter and nptII gene, to form the reference plasmid pMCS. Qualitative detection showed that pMCS is a good positive control for transgenic cotton detection. Real-time PCR detection efficiencies with pMCS as a calibrator ranged from 94.35% to 98.67% for the standard curves of the target genes (R(2)⩾0.998). The relative standard deviation of the mean value for the known sample was 11.95%. These results indicate that the strategy of using the pMCS plasmid as a reference material is feasible and reliable for the detection of transgenic cotton. Therefore, this plasmid can serve as a useful reference tool for qualitative and quantitative detection of single or stacked trait transgenic cotton, thus paving the way for the identification of various products containing components of transgenic cotton. Copyright © 2014 Elsevier Inc. All rights reserved.

Genetically engineered livestock for agriculture: a generation after the first transgenic animal research conference.

PubMed

Murray, James D; Maga, Elizabeth A

2016-06-01

At the time of the first Transgenic Animal Research Conference, the lack of knowledge about promoter, enhancer and coding regions of genes of interest greatly hampered our efforts to create transgenes that would express appropriately in livestock. Additionally, we were limited to gene insertion by pronuclear microinjection. As predicted then, widespread genome sequencing efforts and technological advancements have profoundly altered what we can do. There have been many developments in technology to create transgenic animals since we first met at Granlibakken in 1997, including the advent of somatic cell nuclear transfer-based cloning and gene editing. We can now create new transgenes that will express when and where we want and can target precisely in the genome where we want to make a change or insert a transgene. With the large number of sequenced genomes, we have unprecedented access to sequence information including, control regions, coding regions, and known allelic variants. These technological developments have ushered in new and renewed enthusiasm for the production of transgenic animals among scientists and animal agriculturalists around the world, both for the production of more relevant biomedical research models as well as for agricultural applications. However, even though great advancements have been made in our ability to control gene expression and target genetic changes in our animals, there still are no genetically engineered animal products on the market for food. World-wide there has been a failure of the regulatory processes to effectively move forward. Estimates suggest the world will need to increase our current food production 70 % by 2050; that is we will have to produce the total amount of food each year that has been consumed by mankind over the past 500 years. The combination of transgenic animal technology and gene editing will become increasingly more important tools to help feed the world. However, to date the practical benefits of these technologies have not yet reached consumers in any country and in the absence of predictable, science-based regulatory programs it is unlikely that the benefits will be realized in the short to medium term.

Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests

PubMed Central

Yarasi, Bharathi; Sadumpati, Vijayakumar; Immanni, China Pasalu; Vudem, Dasavantha Reddy; Khareedu, Venkateswara Rao

2008-01-01

Background Rice (Oryza sativa) productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which ~21% is attributed to the attack of insect pests. In this paper we report the isolation, cloning and characterization of Allium sativum leaf agglutinin (asal) gene, and its expression in elite indica rice cultivars using Agrobacterium-mediated genetic transformation method. The stable transgenic lines, expressing ASAL, showed explicit resistance against major sap-sucking pests. Results Allium sativum leaf lectin gene (asal), coding for mannose binding homodimeric protein (ASAL) from garlic plants, has been isolated and introduced into elite indica rice cultivars susceptible to sap-sucking insects, viz., brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH). Embryogenic calli of rice were co-cultivated with Agrobacterium harbouring pSB111 super-binary vector comprising garlic lectin gene asal along with the herbicide resistance gene bar, both under the control of CaMV35S promoter. PCR and Southern blot analyses confirmed stable integration of transgenes into the genomes of rice plants. Northern and western blot analyses revealed expression of ASAL in different transgenic rice lines. In primary transformants, the level of ASAL protein, as estimated by enzyme-linked immunosorbent assay, varied between 0.74% and 1.45% of the total soluble proteins. In planta insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on BPH, GLH and WBPH insects, as evidenced by significant decreases in the survival, development and fecundity of the insects. Conclusion In planta insect bioassays were carried out on asal transgenic rice lines employing standard screening techniques followed in conventional breeding for selection of insect resistant plants. The ASAL expressing rice plants, bestowed with high entomotoxic effects, imparted appreciable resistance against three major sap-sucking insects. Our results amply demonstrate that transgenic indica rice harbouring asal exhibit surpassing resistance against BPH, GLH and WBPH insects. The prototypic asal transgenic rice lines appear promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding. PMID:18854007

Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests.

PubMed

Yarasi, Bharathi; Sadumpati, Vijayakumar; Immanni, China Pasalu; Vudem, Dasavantha Reddy; Khareedu, Venkateswara Rao

2008-10-14

Rice (Oryza sativa) productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which approximately 21% is attributed to the attack of insect pests. In this paper we report the isolation, cloning and characterization of Allium sativum leaf agglutinin (asal) gene, and its expression in elite indica rice cultivars using Agrobacterium-mediated genetic transformation method. The stable transgenic lines, expressing ASAL, showed explicit resistance against major sap-sucking pests. Allium sativum leaf lectin gene (asal), coding for mannose binding homodimeric protein (ASAL) from garlic plants, has been isolated and introduced into elite indica rice cultivars susceptible to sap-sucking insects, viz., brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH). Embryogenic calli of rice were co-cultivated with Agrobacterium harbouring pSB111 super-binary vector comprising garlic lectin gene asal along with the herbicide resistance gene bar, both under the control of CaMV35S promoter. PCR and Southern blot analyses confirmed stable integration of transgenes into the genomes of rice plants. Northern and western blot analyses revealed expression of ASAL in different transgenic rice lines. In primary transformants, the level of ASAL protein, as estimated by enzyme-linked immunosorbent assay, varied between 0.74% and 1.45% of the total soluble proteins. In planta insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on BPH, GLH and WBPH insects, as evidenced by significant decreases in the survival, development and fecundity of the insects. In planta insect bioassays were carried out on asal transgenic rice lines employing standard screening techniques followed in conventional breeding for selection of insect resistant plants. The ASAL expressing rice plants, bestowed with high entomotoxic effects, imparted appreciable resistance against three major sap-sucking insects. Our results amply demonstrate that transgenic indica rice harbouring asal exhibit surpassing resistance against BPH, GLH and WBPH insects. The prototypic asal transgenic rice lines appear promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.

Transformation of Arabidopsis with a Brassica SLG/SRK region and ARC1 gene is not sufficient to transfer the self-incompatibility phenotype.

PubMed

Bi, Y M; Brugière, N; Cui, Y; Goring, D R; Rothstein, S J

2000-05-01

Self-incompatibility (SI) promotes outbreeding in flowering plants, and in Brassica SI is genetically controlled by the S locus. Self-incompatible Brassica and self-fertile Arabidopsis belong to the same crucifer family. In addition, a comparative analysis reveals a high degree of microsynteny between the B. campestris S locus and its homologous region in Arabidopsis--with the notable exception that the Brassica SI genes, SLG and SRK, are missing. Brassica ARC1 encodes a component of the SRK signal transduction pathway leading to self-pollen rejection, and no closely related ARC1 homolog has been identified in Arabidopsis. The purpose of the research reported here was to introduce Brassica SI components into Arabidopsis in an attempt to compensate for the missing genes and to investigate whether the SI phenotype can be transferred. Inserts of approximately 40 kb from the fosmid clones F20 and F22, which span the B. napus W1 SLG-SRK region, were cloned into the plant transformation vector pBIBAC2. Transgenic plants were generated that expressed the Brassica SI genes in the flower buds. In addition, the endogenous, SLG-like, gene AtS1 was not co-suppressed by the Brassica SLG transgene. No SI phenotype was observed among the T1 BIBAC2-F20 and BIBAC2-F22 transgenic plants. When the ARC1 gene was transformed into BIBAC2-F20 or BIBAC2-F22 plants, the resulting BIBAC2-F20-ARC1 and BIBAC2-F22-ARC1 plants still set seeds normally, and no rejection response was observed when self-incompatible B. napus W1 pollen was placed on BIBAC2-F20-ARC1 or BIBAC2-F22-ARC1 Arabidopsis stigmas. Taken together, our results suggest that complementing Arabidopsis genome with Brassica SLG, SRK and ARC1 genes is unlikely to be sufficient to transfer the SI phenotype.

Inhibition of a ubiquitously expressed pectin methyl esterase in Solanum tuberosum L. affects plant growth, leaf growth polarity, and ion partitioning.

PubMed

Pilling, J; Willmitzer, L; Bücking, H; Fisahn, J

2004-05-01

Two pectin methyl esterases (PMEs; EC 3.1.1.11) from Solanum tuberosum were isolated and their expression characterised. One partial clone ( pest1) was expressed in leaves and fruit tissue, while pest2 was a functional full-length clone and was expressed ubiquitously, with a preference for aerial organs. Potato plants were transformed with a chimeric antisense construct that was designed to simultaneously inhibit pest1 and pest2 transcript accumulation; however, reduction of mRNA levels was confined to pest2. The decrease in pest2 transcript was accompanied by up to 50% inhibition of total PME activity, which was probably due to the reduction of only one PME isoform. PME inhibition affected plant development as reflected by smaller stem elongation rates of selected transformants when compared with control plants, leading to a reduction in height throughout the entire course of development. Expansion rates of young developing leaves were measured simultaneously by two displacement transducers in the direction of the leaf tip (proximal-distal axis) and in the perpendicular direction (medial-lateral axis). Significant differences in leaf growth patterns were detected between wild-type and transgenic plants. We suggest that these visual phenotypes could be correlated with modifications of ion accumulation and partitioning within the transgenic plants. The ion-binding capacities of cell walls from PME-inhibited plants were specifically modified as they preferentially bound more sodium, but less potassium and calcium. X-ray microanalysis also indicated an increase in the concentration of several ions within the leaf apoplast of transgenic plants. Moreover, quantification of the total content of major cations revealed differences specific for a given element between the leaves of PME-inhibited and wild-type plants. Reduced growth rates might also be due to effects of PME inhibition on pectin metabolism, predominantly illustrated by an accumulation of galacturonic acid over other cell wall components.

Microbial analysis and survey test of gamma-irradiated freeze-dried fruits for patient's food

NASA Astrophysics Data System (ADS)

Park, Jae-Nam; Sung, Nak-Yun; Byun, Eui-Hong; Byun, Eui-Baek; Song, Beom-Seok; Kim, Jae-Hun; Lee, Kyung-A.; Son, Eun-Joo; Lyu, Eun-Soon

2015-06-01

This study examined the microbiological and organoleptic qualities of gamma-irradiated freeze-dried apples, pears, strawberries, pineapples, and grapes, and evaluated the organoleptic acceptability of the sterilized freeze-dried fruits for hospitalized patients. The freeze-dried fruits were gamma-irradiated at 0, 1, 2, 3, 4, 5, 10, 12, and 15 kGy, and their quality was evaluated. Microorganisms were not detected in apples after 1 kGy, in strawberries and pears after 4 kGy, in pineapples after 5 kGy, and in grapes after 12 kGy of gamma irradiation. The overall acceptance score, of the irradiated freeze-dried fruits on a 7-point scale at the sterilization doses was 5.5, 4.2, 4.0, 4.1, and 5.1 points for apples, strawberries, pears, pineapples, and grapes, respectively. The sensory survey of the hospitalized cancer patients (N=102) resulted in scores of 3.8, 3.7, 3.9, 3.9, and 3.7 on a 5-point scale for the gamma-irradiated freeze-dried apples, strawberries, pears, pineapples, and grapes, respectively. The results suggest that freeze-dried fruits can be sterilized with a dose of 5 kGy, except for grapes, which require a dose of 12 kGy, and that the organoleptic quality of the fruits is acceptable to immuno-compromised patients. However, to clarify the microbiological quality and safety of freeze-dried fruits should be verified by plating for both aerobic and anaerobic microorganisms.

Effect of vital bleaching with solutions containing different concentrations of hydrogen peroxide and pineapple extract as an additive on human enamel using reflectance spectrophotometer: An in vitro study

PubMed Central

Vejai Vekaash, Chitra Janardhanan; Kumar Reddy, Tripuravaram Vinay; Venkatesh, Kondas Vijay

2017-01-01

Aim: This study aims to evaluate the color change in human enamel bleached with three different concentrations of hydrogen peroxide, containing pineapple extract as an additive in two different timings, using reflectance spectrophotometer. Background: The study aimed to investigate the bleaching efficacy on natural teeth using natural enzymes. Materials and Methods: Baseline color values of 10 randomly selected artificially stained incisors were obtained. The specimens were divided into three groups of 20 teeth each: Group 1 – 30% hydrogen peroxide, Group II – 20% hydrogen peroxide, and Group III – 10% hydrogen peroxide. One half of the tooth was bleached with hydrogen peroxide, and other was bleached with hydrogen peroxide and pineapple extract for 20 min (Subgroup A) and 10 min (Subgroup B). Statistical Analysis: The results were statistically analyzed using student's t-test. Results: The mean ΔE values of Group IA (31.62 ± 0.9), Group IIA (29.85 ± 1.2), and Group IIIA (28.65 ± 1.2) showed statistically significant higher values when compared to the mean Δ E values of Group 1A (25.02 ± 1.2), Group IIA (22.86 ± 1.1), and Group IIIA (16.56 ± 1.1). Identical results were obtained in Subgroup B. Conclusion: The addition of pineapple extract to hydrogen peroxide resulted in effective bleaching. PMID:29386782

Chemical profile of pineapple cv. Vitória in different maturation stages using electrospray ionization mass spectrometry.

PubMed

Ogawa, Elizângela M; Costa, Helber B; Ventura, José A; Caetano, Luiz Cs; Pinto, Fernanda E; Oliveira, Bruno G; Barroso, Maria Eduarda S; Scherer, Rodrigo; Endringer, Denise C; Romão, Wanderson

2018-02-01

Pineapple is the fruit of Ananas comosus var. comosus plant, being cultivated in tropical areas and has high energy content and nutritional value. Herein, 30 samples of pineapple cv. Vitória were analyzed as a function of the maturation stage (0-5) and their physico-chemical parameters monitored. In addition, negative-ion mode electrospray ionization mass spectrometry [ESI(-)FT-ICR MS] was used to identify and semi-quantify primary and secondary metabolites present in the crude and phenolic extracts of pineapple, respectively. Physico-chemical tests show an increase in the total soluble solids (TSS) values and in the TSS/total titratable acidity ratio as a function of the maturity stage, where a maximum value was observed in stage 3 (¾ of the fruit is yellow, which corresponds to the color of the fruit peel). ESI(-)FT-ICR MS analysis for crude extracts showed the presence mainly of sugars as primary metabolites present in deprotonated molecule form ([M - H] - and [2 M - H] - ions) whereas, for phenolic fractions, 11 compounds were detected, being the most abundant in the third stage of maturation. This behavior was confirmed by quantitative analysis of total polyphenols. ESI-FT-ICR MS was efficient in identifying primary (carbohydrates and organic acids) and secondary metabolites (13 phenolic compounds) presents in the crude and phenolic extract of the samples, respectively. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Characterization of Mexican coriander (Eryngium foetidum) essential oil and its inactivation of Listeria monocytogenes in vitro and during mild thermal pasteurization of pineapple juice.

PubMed

Ngang, Jean J Essia; Nyegue, Maximilienne A; Ndoye, Foe C; Tchuenchieu Kamgain, Alex D; Sado Kamdem, Sylvain L; Lanciotti, Rosalba; Gardini, Fausto; Etoa, François-Xavier

2014-03-01

The aim of this work was to characterize the essential oil (EO) of Eryngium foetidum (EfEO) and assess its activity toward Listeria monocytogenes in broth and during thermal inactivation of the pathogen in pineapple juice. In this respect, EfEO was chemically characterized, and its antilisteria potential in broth as a function of pH, cell load, and EfEO concentration was assessed through a central composite design. Furthermore, the inactivation kinetics of L. monocytogenes in the juice were assessed by combining EfEO and low pasteurization temperatures. A total of 81 compounds were identified from EfEO. The reduction of pH and cell load increased EO activity. The use of only 15 ppm of EfEO during pasteurization of pineapple juice at 60°C reduced the time required for a 4-log reduction in L. monocytogenes CFU/ml by 74.9% (i.e., from 8.5 to 2.1 min) compared with treatment without EfEO. It could be concluded that EfEO activity toward L. monocytogenes increases with the reduction of pH and that it can be used at sublethal concentrations in combination with low temperatures in pineapple juice pasteurization. This study demonstrates that EO-assisted pasteurization is a promising strategy for the reduction of thermal impact during juice production. EfEO is easily available and compatible with many juices and is thus promising for industrial application.

Over-expression of mango (Mangifera indica L.) MiARF2 inhibits root and hypocotyl growth of Arabidopsis.

PubMed

Wu, Bei; Li, Yun-He; Wu, Jian-Yong; Chen, Qi-Zhu; Huang, Xia; Chen, Yun-Feng; Huang, Xue-Lin

2011-06-01

An auxin response factor 2 gene, MiARF2, was cloned in our previous study [1] from the cotyledon section of mango (Mangifera indica L. cv. Zihua) during adventitious root formation, which shares an 84% amino acid sequence similarity to Arabidopsis ARF2. This study was to examine the effects of over-expression of the full-length MiARF2 open reading frame on the root and hypocotyl growth in Arabidopsis. Phenotype analysis showed that the T(3) transgenic lines had about 20-30% reduction in the length of hypocotyls and roots of the seedlings in comparison with the wild-type. The transcription levels of ANT and ARGOS genes which play a role in controlling organ size and cell proliferation in the transgenic seedlings also decreased. Therefore, the inhibited root and hypocotyl growth in the transgenic seedlings may be associated with the down-regulated transcription of ANT and ARGOS by the over-expression of MiARF2. This study also suggests that although MiARF2 only has a single DNA-binding domain (DBD), it can function as other ARF-like proteins containing complete DBD, middle region (MR) and carboxy-terminal dimerization domain (CTD).

Myeloblastic leukemia cells conditionally blocked by myc-estrogen receptor chimeric transgenes for terminal differentiation coupled to growth arrest and apoptosis.

PubMed

Selvakumaran, M; Liebermann, D; Hoffman-Liebermann, B

1993-05-01

Conditional mutants of the myeloblastic leukemic M1 cell line, expressing the chimeric mycer transgene, have been established. It is shown that M1 mycer cells, like M1, undergo terminal differentiation coupled to growth arrest and programmed cell death (apoptosis) after treatment with the physiologic differentiation inducer interleukin-6. However, when beta-estradiol is included in the culture medium, M1 mycer cells respond to differentiation inducers like M1 myc cell lines, where the differentiation program is blocked at an intermediate stage. By manipulating the function of the mycer transgene product, it is shown that there is a 10-hour window during myeloid differentiation, from 30 to 40 hours after the addition of the differentiation inducer, when the terminal differentiation program switches from being dependent on c-myc suppression to becoming c-myc suppression independent, where activation of c-myc has no apparent effect on mature macrophages. M1 mycer cell lines provide a powerful tool to increase our understanding of the role of c-myc in normal myelopoiesis and in leukemogenesis, also providing a strategy to clone c-myc target genes.

Antisense expression of an Arabidopsis ran binding protein renders transgenic roots hypersensitive to auxin and alters auxin-induced root growth and development by arresting mitotic progress

NASA Technical Reports Server (NTRS)

Kim, S. H.; Arnold, D.; Lloyd, A.; Roux, S. J.

2001-01-01

We cloned a cDNA encoding an Arabidopsis Ran binding protein, AtRanBP1c, and generated transgenic Arabidopsis expressing the antisense strand of the AtRanBP1c gene to understand the in vivo functions of the Ran/RanBP signal pathway. The transgenic plants showed enhanced primary root growth but suppressed growth of lateral roots. Auxin significantly increased lateral root initiation and inhibited primary root growth in the transformants at 10 pM, several orders of magnitude lower than required to induce these responses in wild-type roots. This induction was followed by a blockage of mitosis in both newly emerged lateral roots and in the primary root, ultimately resulting in the selective death of cells in the tips of both lateral and primary roots. Given the established role of Ran binding proteins in the transport of proteins into the nucleus, these findings are consistent with a model in which AtRanBP1c plays a key role in the nuclear delivery of proteins that suppress auxin action and that regulate mitotic progress in root tips.

Overexpression of a glyoxalase gene, OsGly I, improves abiotic stress tolerance and grain yield in rice (Oryza sativa L.).

PubMed

Zeng, Zhengming; Xiong, Fangjie; Yu, Xiaohong; Gong, Xiaoping; Luo, Juntao; Jiang, Yudong; Kuang, Haochi; Gao, Bijun; Niu, Xiangli; Liu, Yongsheng

2016-12-01

Glyoxalase I (Gly I) is a component of the glyoxalase system which is involved in the detoxification of methylglyoxal, a byproduct of glycolysis. In the present study, a gene of rice (Oryza sativa L., cv. Nipponbare) encoding Gly I was cloned and characterized. The quantitative real-time PCR analysis indicated that rice Gly I (OsGly I) was ubiquitously expressed in root, stem, leaf, leaf sheath and spikelet with varying abundance. OsGly I was markedly upregulated in response to NaCl, ZnCl 2 and mannitol in rice seedlings. For further functional investigation, OsGly I was overexpressed in rice using Agrobacterium-mediated transformation. Transgenic rice lines exhibited increased glyoxalase enzyme activity, decreased methylglyoxal level and improved tolerance to NaCl, ZnCl 2 and mannitol compared to wild-type plants. Enhancement of stress tolerance in transgenic lines was associated with reduction of malondialdehyde content which was derived from cellular lipid peroxidation. In addition, the OsGly I-overexpression transgenic plants performed higher seed setting rate and yield. Collectively, these results indicate the potential of bioengineering the Gly I gene in crops. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Exploiting Multisite Gateway and pENFRUIT plasmid collection for fruit genetic engineering.

PubMed

Estornell, Leandro H; Granell, Antonio; Orzaez, Diego

2012-01-01

MultiSite Gateway cloning techniques based on homologous recombination facilitate the combinatorial assembly of basic genetic pieces (i.e., promoters, CDS, and terminators) into gene expression or gene silencing cassettes. pENFRUIT is a collection of MultiSite Triple Gateway Entry vectors dedicated to genetic engineering in fruits. It comprises a number of fruit-operating promoters as well as C-terminal tags adapted to the Gateway standard. In this way, flanking regulatory/labeling sequences can be easily Gateway-assembled with a given gene of interest for its ectopic expression or silencing in fruits. The resulting gene constructs can be analyzed in stable transgenic plants or in transient expression assays, the latter allowing fast testing of the increasing number of combinations arising from MultiSite methodology. A detailed description of the use of MultiSite cloning methodology for the assembly of pENFRUIT elements is presented.

Cloning and Sequencing of Defective Particles Derived from the Autonomous Parvovirus Minute Virus of Mice for the Construction of Vectors with Minimal cis-Acting Sequences

PubMed Central

Clément, Nathalie; Avalosse, Bernard; El Bakkouri, Karim; Velu, Thierry; Brandenburger, Annick

2001-01-01

The production of wild-type-free stocks of recombinant parvovirus minute virus of mice [MVM(p)] is difficult due to the presence of homologous sequences in vector and helper genomes that cannot easily be eliminated from the overlapping coding sequences. We have therefore cloned and sequenced spontaneously occurring defective particles of MVM(p) with very small genomes to identify the minimal cis-acting sequences required for DNA amplification and virus production. One of them has lost all capsid-coding sequences but is still able to replicate in permissive cells when nonstructural proteins are provided in trans by a helper plasmid. Vectors derived from this particle produce stocks with no detectable wild-type MVM after cotransfection with new, matched, helper plasmids that present no homology downstream from the transgene. PMID:11152501

Cloning and transgenesis in mammals: implications for xenotransplantation.

PubMed

Piedrahita, Jorge A; Mir, Bashir

2004-01-01

Availability of suitable organs for transplantation remains of major concern and projections indicate that the problem will continue to increase. Therefore, alternatives to the use of human organs for transplantation, continue to be explored including use of stem cells, artificial organs, and organs from other species (xenotransplantation). In xenotransplantation, the species of choice remains the pig due to its physiological similarities to humans, reduced costs, ease of manipulation, and reduced ethical concerns to its use. However, in order to develop pig organs that are suitable for xenotransplantation, complex genetic modification need to be undertaken. These modifications require the introduction of precise genetic changes into the pig that can only be accomplished at this time using somatic cell nuclear transfer. We cover in this review advances in transgenic manipulation and cloning in swine and how the development of these two technologies is critical to the eventual utilization of the pig as a human organ donor.

In vitro and in vivo evaluation of the activity of pineapple (Ananas comosus) on Haemonchus contortus in Santa Inês sheep.

PubMed

Domingues, Luciana Ferreira; Giglioti, Rodrigo; Feitosa, Karina Alves; Fantatto, Rafaela Regina; Rabelo, Márcio Dias; de Sena Oliveira, Márcia Cristina; Bechara, Gervásio Henrique; de Oliveira, Gilson Pereira; Barioni Junior, Waldomiro; de Souza Chagas, Ana Carolina

2013-10-18

The development of resistance to anthelmintics has prompted research into alternative methods of controlling intestinal nematodes in ruminants. This study aimed to assess the activity of Ananas comosus on Haemonchus contortus in Santa Inês sheep. The aqueous extract of pineapple skin (AEPS), bromelain from pineapple stems (B4882) and residue from pineapple processing was evaluated in in vitro and in vivo tests. The enzymatic activity of substances was analyzed by the azocasein method. The egg hatch test (EHT) and larval development test (LDT) were performed using the Embrapa2010 isolate of H. contortus. In the in vivo test, 36 sheep artificially infected with H. contortus were divided into six groups: G1: 2 g/kg BW of the aqueous extract administered for three days; G2: 2 g/kg BW of the industrial pineapple residue for 60 days; G3: 180 mg/animal of bromelain in a single dose; G4: negative control I; G5: positive control (levamisole phosphate); and G6: negative control II. The eggs per gram (EPG) in the feces were counted till 28 days after treatment. LC₅₀ and LC₉₀ were obtained by the probit procedure, while the in vivo test results were analyzed by GLM. The aqueous extract in the in vitro and in vivo test, the bromelain and industrial residue presented 0.102, 0.157, 1.864 and 0.048 enzyme units/mL, respectively. In the egg hatch test, the LC₅₀ and LC₉₀ were respectively 31 and 81 mg/mL for the aqueous extract and 0.50 and 2 mg/mL for bromelain. In the larval development test, the LC₅₀ and LC₉₀ were respectively 1.7 and 7.3 mg/mL for the aqueous extract and 0.019 and 0.086 mg/mL for bromelain. In the in vivo test, the general efficacies of the treatments in relation to the negative control were 22.6%, 42.2%, 3.65% and 89% for the aqueous extract, industrial pineapple residue, bromelain and positive control respectively. The transformed EPG values were 3.19 ± 0.59, 3.32 ± 0.25, 2.85 ± 0.66, 3.44 ± 0.50, 2.28 ± 0.93 and 2.75 ± 0.94 for the aqueous extract, industrial residue, bromelain, negative control I, positive control and negative control II respectively. The results for all the treated groups differed significantly (p<0.05) from the positive control, and although the residue presented efficacy of 42.2%, there was no statistical difference (p>0.05) in relation to the negative control. Therefore, both the aqueous extract and bromelain were effective in vitro, but showed reduced anthelmintic efficacy in vivo. For the pineapple residue, the 42.2% in vivo efficacy in reducing the EPG and the possibility of reducing environmental contamination through reuse of industrial residue indicate it can also be useful for control of this parasite. Copyright © 2013 Elsevier B.V. All rights reserved.

The maize WRKY transcription factor ZmWRKY17 negatively regulates salt stress tolerance in transgenic Arabidopsis plants.

PubMed

Cai, Ronghao; Dai, Wei; Zhang, Congsheng; Wang, Yan; Wu, Min; Zhao, Yang; Ma, Qing; Xiang, Yan; Cheng, Beijiu

2017-12-01

We cloned and characterized the ZmWRKY17 gene from maize. Overexpression of ZmWRKY17 in Arabidopsis led to increased sensitivity to salt stress and decreased ABA sensitivity through regulating the expression of some ABA- and stress-responsive genes. The WRKY transcription factors have been reported to function as positive or negative regulators in many different biological processes including plant development, defense regulation and stress response. This study isolated a maize WRKY gene, ZmWRKY17, and characterized its role in tolerance to salt stress by generating transgenic Arabidopsis plants. Expression of the ZmWRKY17 was up-regulated by drought, salt and abscisic acid (ABA) treatments. ZmWRKY17 was localized in the nucleus with no transcriptional activation in yeast. Yeast one-hybrid assay showed that ZmWRKY17 can specifically bind to W-box, and it can activate W-box-dependent transcription in planta. Heterologous overexpression of ZmWRKY17 in Arabidopsis remarkably reduced plant tolerance to salt stress, as determined through physiological analyses of the cotyledons greening rate, root growth, relative electrical leakage and malondialdehyde content. Additionally, ZmWRKY17 transgenic plants showed decreased sensitivity to ABA during seed germination and early seedling growth. Transgenic plants accumulated higher content of ABA than wild-type (WT) plants under NaCl condition. Transcriptome and quantitative real-time PCR analyses revealed that some stress-related genes in transgenic seedlings showed lower expression level than that in the WT when treated with NaCl. Taken together, these results suggest that ZmWRKY17 may act as a negative regulator involved in the salt stress responses through ABA signalling.